Brandon:LabNotes/Project1/2015-9-21: Difference between revisions

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18. Gel Size selection
18. Gel Size selection
*gel size select from 220-1000 bp, follow gel size selection protocol
*do not need to include controls.
===Results===
*total RNA.
*TBU gel after IVT, Guanidine HCl to remove proteins
*qPCR curves
*after adding barcodes, TBE gel
*gel size selection amounts
*gel size selection
*gel size selection validation
===conclusions===
*submitted for sequencing
===multiplex combinatorial cellular indexing adaptation to THS-seq using nXTv2 sequences to assay chromatin accessibility protocol===
*Does tagmentation work fine in the presence of protease inhibitors?
*Using 16% DMF final concentration in solution, any effect on dissociating nuclei? didn't with 10%
*sorting by FACS with DAPI added necessary? just add by dilution?
*using  1/3 beads and 1/2 IVT reagents work fine?
*96 well RNA purification plate for all samples, versus doing beads?
*after 2x stranded cDNA synthesis, no water addition, only 1.8X beads work fine?
*second tagmentation in 20 uL?
*SPRI after second tagmentation eluted in 10 uL? (run beads on gel and see if any product still stuck?)
*reagents etc needed:
**taq5X ~ 1 mL (100)
**clontech MMLV, 1/2 kit, and 1 order of dNTPS (125)
**SPRI beads (~10 mL per run)  (175)
**maxiscript ~1/2 kit per run (150)
**zymo ZR RNA clean and concentrator plate 1 plate, (200)
**qiagen protease, 1/2  (70)
===Before starting protocols===
1. Check if have enough reagents etc for the protocol
*lysis buffer
*tn5059 transposomes
*custom 3' transpson
*IVT reagents
*cells etc
*5X, 2X taq polymerase
*Zymo DNA clean and concentrator
*2. Samples
samples
1. XX
2. XX
===IVT Protocol===
*If need to make more transposome, do first 2 steps. If not goto step 3.
'''Generation of transposomes'''
1. annealing of ME sequence to T7 transposon sequence
a. Make 100 uM stock solution of tranposon and ME only bottom end.
b. Incubate 15 uL of each oligo (100uM) with 20 uL EB buffer at 95C for 2 minutes, then cool to 14C at 0.1 C/s.
c. Oligo's now at 30 uM in 50 uL.
2. Transposome complex generation, run controls!!!
*add the below components into one tube and incubate for 30 minutes at RT
a. Add 1.0 uL of 100% sterile glycerol to tube
b. Add 1.0 uL of annealed transposon to the 100% sterile glycerol and mix well
c. Add 2.0 uL of transposase to well.
*store at -20, is good for a year
3. Prepare samples, lyse cells with lysis buffer
CELL WASHING - (wash cells with PBS)
a. count cells, spin down all cells at 250Xg for 5 minutes
b. wash with 1X PBS, centrifuge 250Xg for 5 minutes, resuspend to 33 cells/uL
c. Count cells again to be sure of cells/uL number
CELL LYSIS
a. dilute Lysis Buffer (LB) to 4X, or as needed (from 10X stock)
b. add 3 uL cells to each well in 96 well plate
c. add 1 uL 4X LB + protease inhibitors to each well, mix briefly
d. transposition ready
LYSIS BUFFER NOTES
*Lysis buffer is: 10ml 10X lysis buffer (LB, 100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2003) in nuclease free H2O.
4. transposition reaction. Add all components and incubate at 37C FOR 30 MINUTES
'''Added in 3 uL cells, then 1 uL 4X LB'''
1.2 uL 5X Custom Tagmentation buffer
4.0 uL lysed cells/pure genomic DNA
1.2 uL T7 transposomes (5X tn5-059, indexed Tsome as listed)
___________
6.4 uL total solution
5. Stop reaction by adding 1 volume (6.5 uL) 40 mM EDTA. Incubate for 15 mins at 37C.
6. Nuclei pooling and recovery
a. Pool all nuclei into one tube
b. Add 8 uL PBS to each well of the 96 well plate, and add to pooled tube
c. Mix pooled tube gently and throughly then aliquot 25 nuclei, 5.25 uL to each well in 96 well plate
        (9600/2016) = 4.76 nuclei/uL;  25/4.76 = 5.25
*sort with FACS? can sort into 10 uL EB buffer.
*In paper: nuclei were first stained with DAPI (Invitrogen) at a final concentration of 3μM and then 25 DAPI-positive nuclei were sorted into each well of a plate with 20μl EB buffer.
7. Bring each well to 10 uL with N-H2O. Add 10 uL 8M guanidine HCl for a total of 20 uL.
**For SPRI beads add 1.8X (36 uL) beads per well and follow beads purification protocol
**'''ELUTE IN 6, leave beads in solution'''
8. Fill in reaction
*Add 1.5 uL of 5X taq polymerase, mix thoroughly. Run at 72C for 3 minutes.
9. Maxiscript (Ambion) T7 Protocol, IVT
*DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification.
a. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U)
    on ice, but keep 10X transcription buffer at room temp
b. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!!
  X    uL  DNA template (list 1 ug)
  1.2  uL  10X Transcription Buffer
  0.6  uL  10 mM ATP
  0.6  uL  10 mM CTP
  0.6  uL  10 mM GTP
  0.6  uL  10 mM UTP
  1.2  uL  T7 Enzyme Mix
  _______________________
  12.3 uL total
b. Incubate reactions at 37C overnight for ~16-19 hours.
10. Clean with ZR-96 RNA Clean & Concentrator, put solution and all beads on columns.
*using beads could be an option?
*elute samples in 9.0 uL of N-H2O
*quanitate/view on TBU gel, only 1 sample?
'''AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO'''
*Amounts of RNA and water to add for MMLV RT (50 ng RNA)
11. single strand synthesis MMLV RT (Clontech)
*Followed protocol for [http://genome-tech.ucsd.edu/LabNotes/index.php/Brandon:Protocols/SMART_MMLV_RT SMART MMLV Reverse Transcriptase]
a. Add 2.5 uL 20 uM (100 ng total) RANDOM HEXAMERS to RNA sample. Bring to final volume of 11.5 uL with
    Nuclease free H2O
b. heat the mixture to 70C for 3 minutes. Immediately cool on ice.
c. Add the following to the reaction.
    4  uL 5X first strand buffer
    2  uL dNTP mix
    2  uL 100 mM DTT
  .5 uL SMART MMLV RT and mix (ADD LAST!!!!!)
  ___________________________
    20 uL total
d. Incuvate first at Room Temperature for 10 minutes. Then incubate at 42C for 60 minutes.
e. Terminate the reaction by heating at 70C for 10 minutes
12. RNase H digestion
*Use .5 Units for 20 uL reaction. Stock is at 5 Units/ul, thus dilute 10X and use 1 uL per reaction.
a. Add 1 uL of 10X diluted Rnase H to the reaction.
b. Incubate for 20 minutes at 37C.
13. Second strand synthesis: Adding sss_scnXTv2 primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since sss_scnXTv2 has a high Tm.
a. Add 2.5 uL of 20 uM sss_scnXTv2 to each reaction.
b. Incubate solutions for 2 minutes at 65C. Cool immediately on ice.
c. Add 5.875 uL taq5X to reactions, Incubate at 72C for 8 minutes
14. Clean DNA with AMPURE beads depending. elute in 10 uL
*'''USED AMPURE DNA BEADS AT 1:1.8X RATIO'''
*add 54 uL beads per reaction
*elute on 10 uL
15. Fragmenting and 3' End tagging with [1X] custom nXTv2_i7 Tn5059 transposome and custom tagmentation buffer D6
3 uL custom tagmentation buffer
X uL cDNA sample
X uL Nuclase free H2O
3 uL prepared 1X T7 transposomes (MAKE SURE TO ADD LAST)
___________
16 uL total solution
Incubate at 55C for 6 minutes, cool briefly on ice after
16. Protease digestion of transposase, protease inactivation
To each tube, add:
4 uL  Qiagen Protease, for 5 uL reaction 1 uL of .5 for .1 AU final. (stock is 5 AU and diluted 10X. want .5 AU/uL final [])
Incubate: 50C 10 minutes, 70C 20 minutes
17. Fill in reaction
*Add 5 uL 5X taq polymerase, run at 72C for 3 minutes.
18. PCR addition of barcodes '''USING NEXTERA XT V2 modified i5 (scT7_S5XX) AND i7 (N7XX) SEQUENCES!!!!!'''
Samples:
1. dlkd
2. fdko
KAPA SYBR FAST qPCR mix until saturation, X35 cycles
1  uL Taq5X
2  uL scT7_S5XX index primer, 2 uL F, (scT7_S5XX) '''USED XXX'''
2  uL nXTv2_i7 index primer, 2 uL R, (i7XX_long or i7XX_orig)
1  uL 25X SYBR green
25 uL DNA template (use half RT reaction)
_____________
30 uL
KAPA SYBR cycles:
72C 3m, 95C 30s, (95C for 10s, 63C for 30s, 72C for 1:30 min) X15, 72C for 3 min, 4C forever
*terminate before curves saturate (usually cycle 6-8)
19. Pool all samples run on 6% PAGE gel, Qubit. Gel Size selection
*gel size select from 220-1000 bp, follow gel size selection protocol
*gel size select from 220-1000 bp, follow gel size selection protocol
*do not need to include controls.
*do not need to include controls.

Revision as of 02:40, 24 September 2015

multiplex combinatorial cellular indexing adaptation to THS-seq using nXTv2 sequence detail and protocol

  • adapting THS-seq to combinatorial method sequences detail, however will use 3 barcodes since initial transposition only inserts 1 barcode, and barcode will be 6 base pairs to save on transposon length.



Original T7R1-Nextera XT V2 TOP (64 bp)

5'- [CATGG][AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’            (T7-nXTv2_i5-1-top)
     \---/  \-/\---------------------/ \--------------------------------/
     spcr T7 rec.  T7 req. forward         read seq.  \-----------------/
                                                              ME


scT7R1-nXTv2_top (74 bp)

5 basepair spacer removed from 5' end
barcode (r5) and {TCCACGCGC} added in, was new sequence added in on combinatorial method, TCCACGC[r5]GC originally

5'- [AATTAATACGACTCACTATAGGGAGA]{TCCACGCGC}(XXXXXX)TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’            (scT7_r5XXX_i5_top)
     \-/\---------------------/                    \--------------------------------/
   T7 rec.  T7 req. forward                            read seq.  \-----------------/
                                                                           ME


Nextera XT V2 Bottom (19 bp with /5Phos/)
 
5'- /5Phos/CTGTCTCTTATACACATCT -3' (P-T7tspn-bot)



T7 description:
                                       +1
         [CATGG][AAT][TAATACGACTCACTATA|G|GGAGA]
[end sequence]   rec.   T7 req -->        [front sequence for optimal binding]



After Annealing:

5'- AATTAATACGACTCACTATAGGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’         (scT7_r5XXX_i5_top)
                                                     3'- TCTACACATATTCTCTGTC/5Phos/-5'   (P-T7tspn-bot)

5'- /5Phos/CTGTCTCTTATACACATCT -3'                                                       (P-T7tspn-bot)
       3'- GACAGAGAATATGTGTAGACTGCGACGGCTGCT[r5]CGCGCACCTAGAGGGATATCACTCAGCATAATTAA -5'  (scT7_r5XXX_i5_top)



Fragments created after transposition:

5'- AATTAATACGACTCACTATAGGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG        NNNNNNNNNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3' 
                                                     3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNNNNNNNNNNN        GACAGAGAATATGTGTAGACTGCGACGGCTGCT[r5]CGCGCACCTAGAGGGATATCACTCAGCATAATTAA -5'



Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom:

5'- NNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'                         5'- AATTAATACGACTCACTATAGGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG        NNNNNNNNNNNNN -3’
3'- NNNNNNNNNNNNN        GACAGAGAATATGTGTAGACTGCGACGGCTGCT[r5]CGCGCACCTAGAGGGATATCACTCAGCATAATTAA -5'                         3'- TCTACACATATTCTCTGTC/5Phos/ ---------NNNN -5' 



After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also)

5'- [AATTAATACGACTCACTATAGGGAGA]TCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN -3'
3'- [TTAATTATGCTGAGTGATATCCCTCT]AGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC (NNNNNNNNN)NNNN -5'

5'- NNNN(NNNNNNNNN) CTGTCTCTTATACACATCTGACGCTGCCGACGA[r5]GCGCGTGGA[TCTCCCTATAGTGAGTCGTATTAATT] -3'
3'- NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT[r5]CGCGCACCT[AGAGGGATATCACTCAGCATAATTAA] -5'



After IVT, All RNA strands will have same 5' end. RNA is also purified after IVT.
First G in GGG is the +1 site for the beginning of RNA synthesis for T7.
for Tm's used used https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator (NEB Tm calculator)
All Tm's calculated using, "taq DNA polymerase", "taq 2X Mastermix"

RNA 5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN -3'



After single strand cDNA synthesis to form cDNA/RNA hybrid

RNA  5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN -3'
cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN -5'



After Rnase H nicking, denatuation of small RNAs? and addition of 5' end primer.
Extension with BST or klenow exo- or DNAP1 or Taq2x (been using taq2x)

DNA  5'- GGGAGATCCACGCGC -3'                                                      (Tm=57C, anneal at 52C) (sss_scnXTv2)
cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN -5'



Now have double stranded DNA, which should be long.

DNA  5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN -3'
cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN -5'
Custom Nextera i7 transposome
i7 adapter
       
5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'        (nXTv2_i7_top)
               3'- TCTACACATATTCTCTGTC/5Phos/ -5' (P-nXTv2_i7_bot)


5'- /5Phos/CTGTCTCTTATACACATCT -3'                (P-nXTv2_i7_bot)
       3’- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5' (nXTv2_i7_top)



After custom nextera tagmentation:

DNA  5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNN--------- /5Phos/CTGTCTCTTATACACATCT -3'
cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN        GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'



After fill in:

DNA  5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'



Amplification (to add i5/i7 barcodes):
for Tm's used used https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator (NEB Tm calculator)
'All Tm's calculated using, "taq DNA polymerase", "taq 2X Mastermix"

5'- AATGATACGGCGACCACCGA -3'                             (i5_PCR_ILAdptr, Tm=59C)
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCCACGCGC -3' (scT7_S5XX, comp region Tm=57C, whole~75C)
                            DNA  5'- GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
                            cDNA 3'- CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                                            (i7XX_orig, comp region Tm=57C, whole~71C)   3'- GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                            (i7XX_long, comp region Tm=59C, whole~71C) 3'- GAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                            (i7_PCR_ILAdptr, Tm=58C)                                           3'- AGCATACGGCAGAAGACGAAC -5'



Final sequencing ready fragment

       nXTv2_i5_index_read ----->                                      nXTv2_read1 ----->                         nXTv2_i7_index_read ----->
5'- AATGATACGGCGACCACCGAGATCTACAC -3'               5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'         5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCCACGCGC[r5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]CCCTCTAGGTGCGCG[r5]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                    3'- AGCAGCCGTCGCAGTCTACACATATTCTCTGTC -5'         3'- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                        <----- r5_index_read                              <----- nXTv2_read2




Sequences to order

scT7_r5XXX_i5_top   Barcoded Transposons
P-T7tspn-bot        5'- /5Phos/CTGTCTCTTATACACATCT -3'
sss_scnXTv2         5'- GGGAGATCCACGCGC -3'
nXTv2_i7_top        5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'
P-nXTv2_i7_bot      5'- /5Phos/CTGTCTCTTATACACATCT -3'

scT7_S5XX           Modified Nextera XT V2 i5 barcode sequences and adaptors
i7XX_long           Extended Nextera XT V2 i7 barcode sequences and adaptors
i5_PCR_ILAdptr      5'- AATGATACGGCGACCACCGA -3'
i7_PCR_ILAdptr      5'- CAAGCAGAAGACGGCATACGA-3'

nXTv2_read1         5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'
nXTv2_i7_index_read 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
nXTv2_i5_index_read 5'- AATGATACGGCGACCACCGAGATCTACAC -3' 
r5_index_read       5'- CTGTCTCTTATACACATCTGACGCTGCCGACGA -3'
nXTv2_read2         5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'


Barcoded scT7_r5XXX_i5_top transposons
74 bp long, using truncated barcodes from Quantitative single-cell RNA-seq with unique molecular identifiers

num.	r5_barcode	r5_smpl_sheet	name	r5_full_sequence_(5'_->_3')
1	TCTAAT	ATTAGA	scT7_r5001_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTAATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
2	ACTCGT	ACGAGT	scT7_r5002_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACTCGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
3	ACGTCA	TGACGT	scT7_r5003_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACGTCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
4	AACGAC	GTCGTT	scT7_r5004_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAACGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
5	TTAGCG	CGCTAA	scT7_r5005_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTAGCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
6	ACAGAC	GTCTGT	scT7_r5006_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACAGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
7	CGTAGG	CCTACG	scT7_r5007_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGTAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
8	GAGCTC	GAGCTC	scT7_r5008_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
9	AATACC	GGTATT	scT7_r5009_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAATACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
10	TCACAC	GTGTGA	scT7_r5010_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCACACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
11	CTGGTA	TACCAG	scT7_r5011_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
12	GATAGG	CCTATC	scT7_r5012_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGATAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
13	TGTCGA	TCGACA	scT7_r5013_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTCGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
14	ACGGGT	ACCCGT	scT7_r5014_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACGGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
15	CAGCAT	ATGCTG	scT7_r5015_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCAGCATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
16	TTGACA	TGTCAA	scT7_r5016_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGACATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
17	TTCAAG	CTTGAA	scT7_r5017_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCAAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
18	GTCCTG	CAGGAC	scT7_r5018_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTCCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
19	CACTGA	TCAGTG	scT7_r5019_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCACTGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
20	TACAGG	CCTGTA	scT7_r5020_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTACAGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
21	ACCTTT	AAAGGT	scT7_r5021_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACCTTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
22	CCGTTC	GAACGG	scT7_r5022_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCGTTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
23	TGTTTG	CAAACA	scT7_r5023_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTTTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
24	GTGGCT	AGCCAC	scT7_r5024_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTGGCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
25	GTACAA	TTGTAC	scT7_r5025_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTACAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
26	CAAAGT	ACTTTG	scT7_r5026_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCAAAGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
27	GCATGA	TCATGC	scT7_r5027_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCATGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
28	CATTAG	CTAATG	scT7_r5028_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCATTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
29	CTCCTT	AAGGAG	scT7_r5029_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
30	ACCCGA	TCGGGT	scT7_r5030_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACCCGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
31	TGCCAC	GTGGCA	scT7_r5031_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGCCACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
32	TACCAA	TTGGTA	scT7_r5032_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTACCAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
33	CGAGGT	ACCTCG	scT7_r5033_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGAGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
34	CCACCA	TGGTGG	scT7_r5034_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCACCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
35	TAATCA	TGATTA	scT7_r5035_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTAATCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
36	AGTCCA	TGGACT	scT7_r5036_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGTCCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
37	CTCAGC	GCTGAG	scT7_r5037_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTCAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
38	TGGACT	AGTCCA	scT7_r5038_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGACTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
39	CAAACC	GGTTTG	scT7_r5039_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCAAACCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
40	ACTCCG	CGGAGT	scT7_r5040_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACTCCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
41	ACCGTG	CACGGT	scT7_r5041_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACCGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
42	AGAACG	CGTTCT	scT7_r5042_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGAACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
43	ATTGAC	GTCAAT	scT7_r5043_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATTGACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
44	GTATCC	GGATAC	scT7_r5044_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTATCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
45	CGGAAA	TTTCCG	scT7_r5045_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGGAAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
46	TTGTGT	ACACAA	scT7_r5046_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGTGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
47	GCACAT	ATGTGC	scT7_r5047_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCACATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
48	GTCATT	AATGAC	scT7_r5048_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTCATTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
49	CCTATA	TATAGG	scT7_r5049_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCCTATATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
50	ACGCTG	CAGCGT	scT7_r5050_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACGCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
51	GATTGT	ACAATC	scT7_r5051_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGATTGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
52	ATGTAG	CTACAT	scT7_r5052_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATGTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
53	CGCTGT	ACAGCG	scT7_r5053_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCTGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
54	AGACTT	AAGTCT	scT7_r5054_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGACTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
55	TGGGCA	TGCCCA	scT7_r5055_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGGCATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
56	ACTAGA	TCTAGT	scT7_r5056_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACTAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
57	ATGGGA	TCCCAT	scT7_r5057_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATGGGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
58	GCGACG	CGTCGC	scT7_r5058_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGCGACGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
59	GGGTAG	CTACCC	scT7_r5059_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGGTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
60	GGTGAA	TTCACC	scT7_r5060_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGGTGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
61	CATCTT	AAGATG	scT7_r5061_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCATCTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
62	ATAATC	GATTAT	scT7_r5062_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATAATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
63	TATGGT	ACCATA	scT7_r5063_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTATGGTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
64	GACTTA	TAAGTC	scT7_r5064_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGACTTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
65	TGATTC	GAATCA	scT7_r5065_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGATTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
66	TAGCGA	TCGCTA	scT7_r5066_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGCGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
67	AACTCC	GGAGTT	scT7_r5067_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAACTCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
68	GAAGTG	CACTTC	scT7_r5068_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGAAGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
69	GAGTAC	GTACTC	scT7_r5069_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGAGTACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
70	CGCAAT	ATTGCG	scT7_r5070_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCAATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
71	CTGGAG	CTCCAG	scT7_r5071_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTGGAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
72	TGGGTT	AACCCA	scT7_r5072_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGGGTTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
73	TCCTAG	CTAGGA	scT7_r5073_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCCTAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
74	TCATGC	GCATGA	scT7_r5074_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCATGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
75	TTCCCT	AGGGAA	scT7_r5075_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTCCCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
76	GTAAAG	CTTTAC	scT7_r5076_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTAAAGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
77	ACAACT	AGTTGT	scT7_r5077_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCACAACTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
78	AGGCAT	ATGCCT	scT7_r5078_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGGCATTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
79	TGTGTC	GACACA	scT7_r5079_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTGTGTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
80	AGCATA	TATGCT	scT7_r5080_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGCATATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
81	AGACGC	GCGTCT	scT7_r5081_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGACGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
82	CTTGCT	AGCAAG	scT7_r5082_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTTGCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
83	TCTGCC	GGCAGA	scT7_r5083_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTCTGCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
84	TAGTCG	CGACTA	scT7_r5084_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTAGTCGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
85	TTGCTG	CAGCAA	scT7_r5085_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTGCTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
86	GACAAC	GTTGTC	scT7_r5086_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGACAACTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
87	ATTCCC	GGGAAT	scT7_r5087_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATTCCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
88	TTAAGC	GCTTAA	scT7_r5088_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCTTAAGCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
89	CGCCTC	GAGGCG	scT7_r5089_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCCTCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
90	CTTATC	GATAAG	scT7_r5090_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCTTATCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
91	CGCTAA	TTAGCG	scT7_r5091_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCCGCTAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
92	AGGTCC	GGACCT	scT7_r5092_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGGTCCTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
93	AAGGTG	CACCTT	scT7_r5093_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAAGGTGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
94	GTTGTA	TACAAC	scT7_r5094_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCGTTGTATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
95	AGAGAA	TTCTCT	scT7_r5095_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCAGAGAATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
96	ATTTGG	CCAAAT	scT7_r5096_i5_top	AATTAATACGACTCACTATAGGGAGATCCACGCGCATTTGGTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
 


Modified Nextera XT V2 i5 barcode sequences and adaptors
same i7 adaptors/barcodes are used as in original Nextera XT V2 system or 2 bp longer ones can be used as listed below

num.	i5_barcode	i5_smplsheet	scT7_i5_name	scT7_i5_full_sequence_(5'_->_3')
1	CTCTCTAT	CTCTCTAT	scT7_S502	AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCCACGCGC
2	TATCCTCT	TATCCTCT	scT7_S503	AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCCACGCGC
3	GTAAGGAG	GTAAGGAG	scT7_S505	AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCCACGCGC
4	ACTGCATA	ACTGCATA	scT7_S506	AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCCACGCGC
5	AAGGAGTA	AAGGAGTA	scT7_S507	AATGATACGGCGACCACCGAGATCTACACAAGGAGTAGGGAGATCCACGCGC
6	CTAAGCCT	CTAAGCCT	scT7_S508	AATGATACGGCGACCACCGAGATCTACACCTAAGCCTGGGAGATCCACGCGC
7	CGTCTAAT	CGTCTAAT	scT7_S510	AATGATACGGCGACCACCGAGATCTACACCGTCTAATGGGAGATCCACGCGC
8	TCTCTCCG	TCTCTCCG	scT7_S511	AATGATACGGCGACCACCGAGATCTACACTCTCTCCGGGGAGATCCACGCGC
9	TCGACTAG	TCGACTAG	scT7_S513	AATGATACGGCGACCACCGAGATCTACACTCGACTAGGGGAGATCCACGCGC
10	TTCTAGCT	TTCTAGCT	scT7_S515	AATGATACGGCGACCACCGAGATCTACACTTCTAGCTGGGAGATCCACGCGC
11	CCTAGAGT	CCTAGAGT	scT7_S516	AATGATACGGCGACCACCGAGATCTACACCCTAGAGTGGGAGATCCACGCGC
12	GCGTAAGA	GCGTAAGA	scT7_S517	AATGATACGGCGACCACCGAGATCTACACGCGTAAGAGGGAGATCCACGCGC
13	CTATTAAG	CTATTAAG	scT7_S518	AATGATACGGCGACCACCGAGATCTACACCTATTAAGGGGAGATCCACGCGC
14	AAGGCTAT	AAGGCTAT	scT7_S520	AATGATACGGCGACCACCGAGATCTACACAAGGCTATGGGAGATCCACGCGC
15	GAGCCTTA	GAGCCTTA	scT7_S521	AATGATACGGCGACCACCGAGATCTACACGAGCCTTAGGGAGATCCACGCGC
16	TTATGCGA	TTATGCGA	scT7_S522	AATGATACGGCGACCACCGAGATCTACACTTATGCGAGGGAGATCCACGCGC


 num.	i7_barcode	i7_name	i7_smplsheet	i7_full_sequence_(5'_->_3')
1	TCGCCTTA	N701_long	TAAGGCGA	CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAG
2	CTAGTACG	N702_long	CGTACTAG	CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAG
3	TTCTGCCT	N703_long	AGGCAGAA	CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAG
4	GCTCAGGA	N704_long	TCCTGAGC	CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAG
5	AGGAGTCC	N705_long	GGACTCCT	CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGGAG
6	CATGCCTA	N706_long	TAGGCATG	CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGGAG
7	GTAGAGAG	N707_long	CTCTCTAC	CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGGAG
8	CAGCCTCG	N710_long	CGAGGCTG	CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGGAG
9	TGCCTCTT	N711_long	AAGAGGCA	CAAGCAGAAGACGGCATACGAGATTGCCTCTTGTCTCGTGGGCTCGGAG
10	TCCTCTAC	N712_long	GTAGAGGA	CAAGCAGAAGACGGCATACGAGATTCCTCTACGTCTCGTGGGCTCGGAG
11	TCATGAGC	N714_long	GCTCATGA	CAAGCAGAAGACGGCATACGAGATTCATGAGCGTCTCGTGGGCTCGGAG
12	CCTGAGAT	N715_long	ATCTCAGG	CAAGCAGAAGACGGCATACGAGATCCTGAGATGTCTCGTGGGCTCGGAG
13	TAGCGAGT	N716_long	ACTCGCTA	CAAGCAGAAGACGGCATACGAGATTAGCGAGTGTCTCGTGGGCTCGGAG
14	GTAGCTCC	N718_long	GGAGCTAC	CAAGCAGAAGACGGCATACGAGATGTAGCTCCGTCTCGTGGGCTCGGAG
15	TACTACGC	N719_long	GCGTAGTA	CAAGCAGAAGACGGCATACGAGATTACTACGCGTCTCGTGGGCTCGGAG
16	AGGCTCCG	N720_long	CGGAGCCT	CAAGCAGAAGACGGCATACGAGATAGGCTCCGGTCTCGTGGGCTCGGAG
17	GCAGCGTA	N721_long	TACGCTGC	CAAGCAGAAGACGGCATACGAGATGCAGCGTAGTCTCGTGGGCTCGGAG
18	CTGCGCAT	N722_long	ATGCGCAG	CAAGCAGAAGACGGCATACGAGATCTGCGCATGTCTCGTGGGCTCGGAG
19	GAGCGCTA	N723_long	TAGCGCTC	CAAGCAGAAGACGGCATACGAGATGAGCGCTAGTCTCGTGGGCTCGGAG
20	CGCTCAGT	N724_long	ACTGAGCG	CAAGCAGAAGACGGCATACGAGATCGCTCAGTGTCTCGTGGGCTCGGAG
21	GTCTTAGG	N726_long	CCTAAGAC	CAAGCAGAAGACGGCATACGAGATGTCTTAGGGTCTCGTGGGCTCGGAG
22	ACTGATCG	N727_long	CGATCAGT	CAAGCAGAAGACGGCATACGAGATACTGATCGGTCTCGTGGGCTCGGAG
23	TAGCTGCA	N728_long	TGCAGCTA	CAAGCAGAAGACGGCATACGAGATTAGCTGCAGTCTCGTGGGCTCGGAG
24	GACGTCGA	N729_long	TCGACGTC	CAAGCAGAAGACGGCATACGAGATGACGTCGAGTCTCGTGGGCTCGGAG

 original Nextera XT V2 i5/i7 barcode sequences and primers

num.	i5_barcode	i5_name	i5_smplsheet	i5_full_sequence_(5'_->_3')
1	CTCTCTAT	S502	CTCTCTAT	AATGATACGGCGACCACCGAGATCTACACCTCTCTATTCGTCGGCAGCGTC
2	TATCCTCT	S503	TATCCTCT	AATGATACGGCGACCACCGAGATCTACACTATCCTCTTCGTCGGCAGCGTC
3	GTAAGGAG	S505	GTAAGGAG	AATGATACGGCGACCACCGAGATCTACACGTAAGGAGTCGTCGGCAGCGTC
4	ACTGCATA	S506	ACTGCATA	AATGATACGGCGACCACCGAGATCTACACACTGCATATCGTCGGCAGCGTC
5	AAGGAGTA	S507	AAGGAGTA	AATGATACGGCGACCACCGAGATCTACACAAGGAGTATCGTCGGCAGCGTC
6	CTAAGCCT	S508	CTAAGCCT	AATGATACGGCGACCACCGAGATCTACACCTAAGCCTTCGTCGGCAGCGTC
7	CGTCTAAT	S510	CGTCTAAT	AATGATACGGCGACCACCGAGATCTACACCGTCTAATTCGTCGGCAGCGTC
8	TCTCTCCG	S511	TCTCTCCG	AATGATACGGCGACCACCGAGATCTACACTCTCTCCGTCGTCGGCAGCGTC
9	TCGACTAG	S513	TCGACTAG	AATGATACGGCGACCACCGAGATCTACACTCGACTAGTCGTCGGCAGCGTC
10	TTCTAGCT	S515	TTCTAGCT	AATGATACGGCGACCACCGAGATCTACACTTCTAGCTTCGTCGGCAGCGTC
11	CCTAGAGT	S516	CCTAGAGT	AATGATACGGCGACCACCGAGATCTACACCCTAGAGTTCGTCGGCAGCGTC
12	GCGTAAGA	S517	GCGTAAGA	AATGATACGGCGACCACCGAGATCTACACGCGTAAGATCGTCGGCAGCGTC
13	CTATTAAG	S518	CTATTAAG	AATGATACGGCGACCACCGAGATCTACACCTATTAAGTCGTCGGCAGCGTC
14	AAGGCTAT	S520	AAGGCTAT	AATGATACGGCGACCACCGAGATCTACACAAGGCTATTCGTCGGCAGCGTC
15	GAGCCTTA	S521	GAGCCTTA	AATGATACGGCGACCACCGAGATCTACACGAGCCTTATCGTCGGCAGCGTC
16	TTATGCGA	S522	TTATGCGA	AATGATACGGCGACCACCGAGATCTACACTTATGCGATCGTCGGCAGCGTC
				
				
num.	i7_barcode	i7_name	i7_smplsheet	i7_full_sequence_(5'_->_3')
1	TCGCCTTA	N701	TAAGGCGA	CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGG
2	CTAGTACG	N702	CGTACTAG	CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGG
3	TTCTGCCT	N703	AGGCAGAA	CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGG
4	GCTCAGGA	N704	TCCTGAGC	CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGG
5	AGGAGTCC	N705	GGACTCCT	CAAGCAGAAGACGGCATACGAGATAGGAGTCCGTCTCGTGGGCTCGG
6	CATGCCTA	N706	TAGGCATG	CAAGCAGAAGACGGCATACGAGATCATGCCTAGTCTCGTGGGCTCGG
7	GTAGAGAG	N707	CTCTCTAC	CAAGCAGAAGACGGCATACGAGATGTAGAGAGGTCTCGTGGGCTCGG
8	CAGCCTCG	N710	CGAGGCTG	CAAGCAGAAGACGGCATACGAGATCAGCCTCGGTCTCGTGGGCTCGG
9	TGCCTCTT	N711	AAGAGGCA	CAAGCAGAAGACGGCATACGAGATTGCCTCTTGTCTCGTGGGCTCGG
10	TCCTCTAC	N712	GTAGAGGA	CAAGCAGAAGACGGCATACGAGATTCCTCTACGTCTCGTGGGCTCGG
11	TCATGAGC	N714	GCTCATGA	CAAGCAGAAGACGGCATACGAGATTCATGAGCGTCTCGTGGGCTCGG
12	CCTGAGAT	N715	ATCTCAGG	CAAGCAGAAGACGGCATACGAGATCCTGAGATGTCTCGTGGGCTCGG
13	TAGCGAGT	N716	ACTCGCTA	CAAGCAGAAGACGGCATACGAGATTAGCGAGTGTCTCGTGGGCTCGG
14	GTAGCTCC	N718	GGAGCTAC	CAAGCAGAAGACGGCATACGAGATGTAGCTCCGTCTCGTGGGCTCGG
15	TACTACGC	N719	GCGTAGTA	CAAGCAGAAGACGGCATACGAGATTACTACGCGTCTCGTGGGCTCGG
16	AGGCTCCG	N720	CGGAGCCT	CAAGCAGAAGACGGCATACGAGATAGGCTCCGGTCTCGTGGGCTCGG
17	GCAGCGTA	N721	TACGCTGC	CAAGCAGAAGACGGCATACGAGATGCAGCGTAGTCTCGTGGGCTCGG
18	CTGCGCAT	N722	ATGCGCAG	CAAGCAGAAGACGGCATACGAGATCTGCGCATGTCTCGTGGGCTCGG
19	GAGCGCTA	N723	TAGCGCTC	CAAGCAGAAGACGGCATACGAGATGAGCGCTAGTCTCGTGGGCTCGG
20	CGCTCAGT	N724	ACTGAGCG	CAAGCAGAAGACGGCATACGAGATCGCTCAGTGTCTCGTGGGCTCGG
21	GTCTTAGG	N726	CCTAAGAC	CAAGCAGAAGACGGCATACGAGATGTCTTAGGGTCTCGTGGGCTCGG
22	ACTGATCG	N727	CGATCAGT	CAAGCAGAAGACGGCATACGAGATACTGATCGGTCTCGTGGGCTCGG
23	TAGCTGCA	N728	TGCAGCTA	CAAGCAGAAGACGGCATACGAGATTAGCTGCAGTCTCGTGGGCTCGG
24	GACGTCGA	N729	TCGACGTC	CAAGCAGAAGACGGCATACGAGATGACGTCGAGTCTCGTGGGCTCGG



Validation of THS-seq barcoded transposons using Nextera XT v2 sequences for multiplex combinatorial indexing

  • designed scT7_r5XXX_i5_top transposon so it can be used used with 6 base pair barcodes. 96 barcoded transposons made. Will first test with tn5059 transposomes from Trina for validation the transposon is working and that fragments can be sequenced on MiSeq/HiSeq. Then will proceed to order the rest of the transposons and use with combinatorial sequencing with THS-seq for single cells.


  • There is slight modification to MiSeq protocol, custom primers will need to be used and Read2 will instead be a 6 basepair index reading using r5_index_read. Standard Nextera XT v2 oligos i5 index, i7 index and read1 are used for sequencing.


Before starting protocols

1. Check if have enough reagents etc for the protocol

  • lysis buffer
  • tn5059 transposomes
  • custom 3' transpson
  • IVT reagents
  • cells etc
  • 5X, 2X taq polymerase
  • Zymo DNA clean and concentrator


  • 2. Samples
samples
1. scT7_i502-i701, scT7_r5001_i5_top, 5X tn5-059, 500 cells rep1
2. scT7_i503-i701, scT7_r5001_i5_top, 5X tn5-059, 500 cells rep2
3. scT7_i505-i701, scT7_r5002_i5_top, 5X tn5-059, 500 cells rep1
4. scT7_i502-i702, scT7_r5002_i5_top, 5X tn5-059, 500 cells rep2
5. scT7_i503-i702, scT7_r5001_i5_top, 5X tn5-059, 6 ng pure DNA
6. scT7_i505-i702, scT7_r5001_i5_top, 5X tn5-059, NTC

IVT Protocol

  • If need to make more transposome, do first 2 steps. If not goto step 3.

Generation of transposomes

1. annealing of ME sequence to T7 transposon sequence

a. Make 100 uM stock solution of tranposon and ME only bottom end.
b. Incubate 15 uL of each oligo (100uM) with 20 uL EB buffer at 95C for 2 minutes, then cool to 14C at 0.1 C/s.
c. Oligo's now at 30 uM in 50 uL.


2. Transposome complex generation, run controls!!!

  • add the below components into one tube and incubate for 30 minutes at RT
a. Add 1.0 uL of 100% sterile glycerol to tube
b. Add 1.0 uL of annealed transposon to the 100% sterile glycerol and mix well
c. Add 2.0 uL of transposase to well.
  • store at -20, is good for a year


3. Prepare samples, lyse cells with lysis buffer

CELL WASHING - (wash cells with PBS)

a. count cells, spin down all cells at 250Xg for 5 minutes
b. wash with 1X PBS, centrifuge 250Xg for 5 minutes, resuspend to 1000 cells/uL
c. Count cells again to be sure of cells/uL amount.

CELL LYSIS

a. dilute Lysis Buffer (LB) to 2X, or as needed (from 10X stock)
b. add 1 uL LB to sample tubes 
c. add 1 uL cells to sample tubes with LB in it, mix briefly (mineral oil optional)
d. transposition ready


LYSIS BUFFER NOTES

  • Lysis buffer is: 10ml 10X lysis buffer (LB, 100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2003) in nuclease free H2O.


4. transposition reaction. Add all components and incubate at 37C FOR 30 MINUTES

1.0 uL 5X Custom Tagmentation buffer
2.0 uL lysed cells/pure genomic DNA
1.0 uL T7 transposomes (5X tn5-059, indexed Tsome as listed)
1.0 uL N-H2O
___________
5.0 uL total solution


5. Bring to 15 uL with N-H2O. Add 15 uL 8M guanidine HCl for a total of 30 uL.

    • For SPRI beads add 1.8X (54 uL) beads per sample. and follow beads protocol
    • ELUTE IN 9.6, leave beads in solution


6. Fill in reaction

  • Add 2.4 uL of 5X taq polymerase, mix thoroughly. Run at 72C for 3 minutes.


7. Maxiscript (Ambion) T7 Protocol, IVT

  • DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification.
a. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U) 
   on ice, but keep 10X transcription buffer at room temp

b. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!!
  X   uL   DNA template (list 1 ug)
  2   uL   10X Transcription Buffer
  1   uL   10 mM ATP
  1   uL   10 mM CTP
  1   uL   10 mM GTP
  1   uL   10 mM UTP
  2   uL   T7 Enzyme Mix
 _______________________
  20 uL total 


b. Incubate reactions at 37C overnight for ~16-19 hours. (>10 uM limiting nucleotide)


8. Clean with Zymo RNA clean and concentrator, put solution and all beads on column.

  • elute samples in 10 uL of N-H2O
  • quanitate/view on TBU gel.



AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO

sample list
1. dkljd
2. ldkf

  • Amounts of RNA and water to add for MMLV RT (50 ng RNA)


9. single strand synthesis MMLV RT (Clontech)


a. Add 2.5 uL 20 uM (100 ng total) RANDOM HEXAMERS to RNA sample. Bring to final volume of 11.5 uL with
   Nuclease free H2O

b. heat the mixture to 70C for 3 minutes.  Immediately cool on ice.

c. Add the following to the reaction.
   4  uL 5X first strand buffer
   2  uL dNTP mix
   2  uL 100 mM DTT
  .5 uL SMART MMLV RT and mix (ADD LAST!!!!!)
  ___________________________
   20 uL total

d. Incuvate first at Room Temperature for 10 minutes. Then incubate at 42C for 60 minutes.

e. Terminate the reaction by heating at 70C for 10 minutes


10. RNase H digestion

  • Use .5 Units for 20 uL reaction. Stock is at 5 Units/ul, thus dilute 10X and use 1 uL per reaction.

a. Add 1 uL of 10X diluted Rnase H to the reaction.

b. Incubate for 20 minutes at 37C.


11. Second strand synthesis: Adding sss_scnXTv2 primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since sss_scnXTv2 has a high Tm.

a. Add 2.5 uL of 20 uM sss_scnXTv2 to each reaction.

b. Incubate solutions for 2 minutes at 65C. Cool immediately on ice.

c. Add 5.875 uL taq5X to reactions, Incubate at 72C for 8 minutes


12. Clean DNA with AMPURE beads depending. elute in 20 uL and concentrate.

  • USED AMPURE DNA BEADS AT 1:1.8X RATIO
  • add 45 uL N-H2O
  • add 135 uL beads per reaction
  • elute on 20 uL


13. After elution in 20 uL, use vacuum centrifuge commentator to concentrate sample size to ~4-5 uL per sample.

  • Also ~4-5 uL per sample is preferred for transposition, which has always been performed in low volume solutions.

RAN FOR 16 MINUTES


14. Fragmenting and 3' End tagging with [1X] custom nXTv2_i7 Tn5059 transposome and custom tagmentation buffer D6

1 uL custom tagmentation buffer
X uL cDNA sample
X uL Nuclase free H2O
1 uL prepared 1X T7 transposomes (MAKE SURE TO ADD LAST)
___________
5 uL total solution

Incubate at 55C for 6 minutes, cool briefly on ice after


15. Protease digestion of transposase, protease inactivation

To each tube, add:
1 uL  Qiagen Protease, for 5 uL reaction 1 uL of .5 for .1 AU final. (stock is 5 AU and diluted 10X. want .5 AU/uL final [])
Incubate: 50C 10 minutes, 70C 20 minutes


16. Fill in reaction

  • Add 6 uL 2X taq polymerase, run at 72C for 3 minutes.


17. PCR addition of barcodes USING NEXTERA XT V2 modified i5 (scT7_S5XX) AND i7 (N7XX) SEQUENCES!!!!!

Samples:
1. dlkd
2. fdko
KAPA SYBR FAST qPCR mix until saturation, X35 cycles

9  uL Taq2X
2  uL scT7_S5XX index primer, 2 uL F, (scT7_S5XX) USED XXX
2  uL nXTv2_i7 index primer, 2 uL R, (i7XX_long or i7XX_orig)
4  uL H2O
1  uL 25X SYBR green
12 uL DNA template (use half RT reaction)
_____________
30 uL

KAPA SYBR cycles:
72C 3m, 95C 30s, (95C for 10s, 63C for 30s, 72C for 1:30 min) X15, 72C for 3 min, 4C forever

  • terminate before curves saturate (usually cycle 6-8)


18. Gel Size selection

  • gel size select from 220-1000 bp, follow gel size selection protocol
  • do not need to include controls.



Results

  • total RNA.


  • TBU gel after IVT, Guanidine HCl to remove proteins


  • qPCR curves


  • after adding barcodes, TBE gel


  • gel size selection amounts


  • gel size selection


  • gel size selection validation


conclusions

  • submitted for sequencing



multiplex combinatorial cellular indexing adaptation to THS-seq using nXTv2 sequences to assay chromatin accessibility protocol

  • Does tagmentation work fine in the presence of protease inhibitors?
  • Using 16% DMF final concentration in solution, any effect on dissociating nuclei? didn't with 10%
  • sorting by FACS with DAPI added necessary? just add by dilution?
  • using 1/3 beads and 1/2 IVT reagents work fine?
  • 96 well RNA purification plate for all samples, versus doing beads?
  • after 2x stranded cDNA synthesis, no water addition, only 1.8X beads work fine?
  • second tagmentation in 20 uL?
  • SPRI after second tagmentation eluted in 10 uL? (run beads on gel and see if any product still stuck?)
  • reagents etc needed:
    • taq5X ~ 1 mL (100)
    • clontech MMLV, 1/2 kit, and 1 order of dNTPS (125)
    • SPRI beads (~10 mL per run) (175)
    • maxiscript ~1/2 kit per run (150)
    • zymo ZR RNA clean and concentrator plate 1 plate, (200)
    • qiagen protease, 1/2 (70)


Before starting protocols

1. Check if have enough reagents etc for the protocol

  • lysis buffer
  • tn5059 transposomes
  • custom 3' transpson
  • IVT reagents
  • cells etc
  • 5X, 2X taq polymerase
  • Zymo DNA clean and concentrator


  • 2. Samples
samples
1. XX
2. XX

IVT Protocol

  • If need to make more transposome, do first 2 steps. If not goto step 3.

Generation of transposomes

1. annealing of ME sequence to T7 transposon sequence

a. Make 100 uM stock solution of tranposon and ME only bottom end.
b. Incubate 15 uL of each oligo (100uM) with 20 uL EB buffer at 95C for 2 minutes, then cool to 14C at 0.1 C/s.
c. Oligo's now at 30 uM in 50 uL.


2. Transposome complex generation, run controls!!!

  • add the below components into one tube and incubate for 30 minutes at RT
a. Add 1.0 uL of 100% sterile glycerol to tube
b. Add 1.0 uL of annealed transposon to the 100% sterile glycerol and mix well
c. Add 2.0 uL of transposase to well.
  • store at -20, is good for a year


3. Prepare samples, lyse cells with lysis buffer

CELL WASHING - (wash cells with PBS)

a. count cells, spin down all cells at 250Xg for 5 minutes
b. wash with 1X PBS, centrifuge 250Xg for 5 minutes, resuspend to 33 cells/uL
c. Count cells again to be sure of cells/uL number

CELL LYSIS

a. dilute Lysis Buffer (LB) to 4X, or as needed (from 10X stock)
b. add 3 uL cells to each well in 96 well plate
c. add 1 uL 4X LB + protease inhibitors to each well, mix briefly
d. transposition ready


LYSIS BUFFER NOTES

  • Lysis buffer is: 10ml 10X lysis buffer (LB, 100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2003) in nuclease free H2O.


4. transposition reaction. Add all components and incubate at 37C FOR 30 MINUTES Added in 3 uL cells, then 1 uL 4X LB

1.2 uL 5X Custom Tagmentation buffer
4.0 uL lysed cells/pure genomic DNA
1.2 uL T7 transposomes (5X tn5-059, indexed Tsome as listed)
___________
6.4 uL total solution


5. Stop reaction by adding 1 volume (6.5 uL) 40 mM EDTA. Incubate for 15 mins at 37C.


6. Nuclei pooling and recovery

a. Pool all nuclei into one tube
b. Add 8 uL PBS to each well of the 96 well plate, and add to pooled tube
c. Mix pooled tube gently and throughly then aliquot 25 nuclei, 5.25 uL to each well in 96 well plate
       (9600/2016) = 4.76 nuclei/uL;  25/4.76 = 5.25
  • sort with FACS? can sort into 10 uL EB buffer.
  • In paper: nuclei were first stained with DAPI (Invitrogen) at a final concentration of 3μM and then 25 DAPI-positive nuclei were sorted into each well of a plate with 20μl EB buffer.


7. Bring each well to 10 uL with N-H2O. Add 10 uL 8M guanidine HCl for a total of 20 uL.

    • For SPRI beads add 1.8X (36 uL) beads per well and follow beads purification protocol
    • ELUTE IN 6, leave beads in solution


8. Fill in reaction

  • Add 1.5 uL of 5X taq polymerase, mix thoroughly. Run at 72C for 3 minutes.


9. Maxiscript (Ambion) T7 Protocol, IVT

  • DNA from PCR can be used directly in the MAXIscript Kit without any pretreatment or purification.
a. Thaw 10X Transcription Buffer and ribonucleotide solutions. Store the ribonucleotides (A, C, G, U) 
   on ice, but keep 10X transcription buffer at room temp

b. Assemble reaction mixture at room temperature, ADD IN ORDER AND MIX THOROUGHLY!!!!
  X     uL   DNA template (list 1 ug)
  1.2   uL   10X Transcription Buffer
  0.6   uL   10 mM ATP
  0.6   uL   10 mM CTP
  0.6   uL   10 mM GTP
  0.6   uL   10 mM UTP
  1.2   uL   T7 Enzyme Mix
 _______________________
  12.3 uL total 


b. Incubate reactions at 37C overnight for ~16-19 hours.


10. Clean with ZR-96 RNA Clean & Concentrator, put solution and all beads on columns.

  • using beads could be an option?
  • elute samples in 9.0 uL of N-H2O
  • quanitate/view on TBU gel, only 1 sample?



AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO


  • Amounts of RNA and water to add for MMLV RT (50 ng RNA)


11. single strand synthesis MMLV RT (Clontech)


a. Add 2.5 uL 20 uM (100 ng total) RANDOM HEXAMERS to RNA sample. Bring to final volume of 11.5 uL with
   Nuclease free H2O

b. heat the mixture to 70C for 3 minutes. Immediately cool on ice.

c. Add the following to the reaction.
   4  uL 5X first strand buffer
   2  uL dNTP mix
   2  uL 100 mM DTT
  .5 uL SMART MMLV RT and mix (ADD LAST!!!!!)
  ___________________________
   20 uL total

d. Incuvate first at Room Temperature for 10 minutes. Then incubate at 42C for 60 minutes.

e. Terminate the reaction by heating at 70C for 10 minutes


12. RNase H digestion

  • Use .5 Units for 20 uL reaction. Stock is at 5 Units/ul, thus dilute 10X and use 1 uL per reaction.

a. Add 1 uL of 10X diluted Rnase H to the reaction.

b. Incubate for 20 minutes at 37C.


13. Second strand synthesis: Adding sss_scnXTv2 primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since sss_scnXTv2 has a high Tm.

a. Add 2.5 uL of 20 uM sss_scnXTv2 to each reaction.

b. Incubate solutions for 2 minutes at 65C. Cool immediately on ice.

c. Add 5.875 uL taq5X to reactions, Incubate at 72C for 8 minutes


14. Clean DNA with AMPURE beads depending. elute in 10 uL

  • USED AMPURE DNA BEADS AT 1:1.8X RATIO
  • add 54 uL beads per reaction
  • elute on 10 uL


15. Fragmenting and 3' End tagging with [1X] custom nXTv2_i7 Tn5059 transposome and custom tagmentation buffer D6

3 uL custom tagmentation buffer
X uL cDNA sample
X uL Nuclase free H2O
3 uL prepared 1X T7 transposomes (MAKE SURE TO ADD LAST)
___________
16 uL total solution

Incubate at 55C for 6 minutes, cool briefly on ice after


16. Protease digestion of transposase, protease inactivation

To each tube, add:
4 uL  Qiagen Protease, for 5 uL reaction 1 uL of .5 for .1 AU final. (stock is 5 AU and diluted 10X. want .5 AU/uL final [])
Incubate: 50C 10 minutes, 70C 20 minutes


17. Fill in reaction

  • Add 5 uL 5X taq polymerase, run at 72C for 3 minutes.


18. PCR addition of barcodes USING NEXTERA XT V2 modified i5 (scT7_S5XX) AND i7 (N7XX) SEQUENCES!!!!!

Samples:
1. dlkd
2. fdko
KAPA SYBR FAST qPCR mix until saturation, X35 cycles

1  uL Taq5X
2  uL scT7_S5XX index primer, 2 uL F, (scT7_S5XX) USED XXX
2  uL nXTv2_i7 index primer, 2 uL R, (i7XX_long or i7XX_orig)
1  uL 25X SYBR green
25 uL DNA template (use half RT reaction)
_____________
30 uL

KAPA SYBR cycles:
72C 3m, 95C 30s, (95C for 10s, 63C for 30s, 72C for 1:30 min) X15, 72C for 3 min, 4C forever

  • terminate before curves saturate (usually cycle 6-8)


19. Pool all samples run on 6% PAGE gel, Qubit. Gel Size selection

  • gel size select from 220-1000 bp, follow gel size selection protocol
  • do not need to include controls.



Results

  • total RNA.


  • TBU gel after IVT, Guanidine HCl to remove proteins


  • qPCR curves


  • after adding barcodes, TBE gel


  • gel size selection amounts


  • gel size selection


  • gel size selection validation


conclusions

  • submitted for sequencing