Brandon:LabNotes/Project1/2015-8-3

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Update of THS-seq protocol sequences to use Nextera XT V2 sequences



  • Called illumina and they said it is either sequencer not being cleaned right, or concentrations are off, or sequences in library generation protocol are not compatible. Considering currently old nextera sequences are being used from 5 years ago, and all current systems illumina sequencing systems do not use them, then maybe that is the problem.



Sequences information, Using T7-nXTv2_i5-1-top and T7-nXTv2_i5-2-top

Sequences:
ORIGINAL T7tspn-top2: (69 bp)
5'- [CATGAGA][TTAATACGACTCACTATAG][GGAGA][TCCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’ (T7tspn-top2)
      spcr      T7 forward    required spcr   read sequence  spcr       ME 

NEXTERA XT V2 R1 TOP (64 bp)
5'- [CATGG][AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’            (T7-nXTv2_i5-1-top)
     \---/  \-/\---------------------/ \--------------------------------/
     spcr T7 rec.  T7 req. forward         read seq.  \-----------------/
                                                              ME  
                                                                                 
NEXTERA XT V2 R1 TOP + EXTRA (69 bp)
5'- [CATGG][AATTAATACGACTCACTATAGGGAGA][CTCCT][TCGTCGGCAGCGTC[AGATGTGTATAAGAGACAG] -3’    (T7-nXTv2_i5-2-top)    
     \---/  \-/\---------------------/  \---/  \--------------------------------/
     spcr T7 rec.  T7 req. forward      spcr      read seq.   \-----------------/
                                                                     ME  


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'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’       (T7-nXTv2-1-top)
                                                    3'- TCTACACATATTCTCTGTC [Phos]-5' (P-T7tspn-bot)


5'- [phos] CTGTCTCTTATACACATCT -3'
      3’-  GACAGAGAATATGTGTAGACTGCGACGGCTGCTAGAGGGATATCACTCAGCATAATTAAGGTAC -5’


Fragments created after transposition:

5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG    NNNNNNNNNN--------- 5P/CTGTCTCTTATACACATCT -3'
                                             3'- TCTACACATATTCTCTGTC/5P ---------NNNNNNNNNN    GACAGAGAATATGTGTAGACTGCGACGGCTGCTAGAGGGATATCACTCAGCATAATTAAGGTAC -5’


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

5'- NNN--------- 5P/CTGTCTCTTATACACATCT -3'           5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG    NNNNNNNNNNNN -3'
3'- NNNNNNNNNNNN    GACAGAGAATATGTGTAGACTGCGACGGCTGCTAGAGGGATATCACTCAGCATAATTAAGGTAC -5’           3'- TCTACACATATTCTCTGTC/5P ---------NNN -5'


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

5'- CATGG[AATTAATACGACTCACTATAGGGAGA]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN -3'
3'- GTACC[TTAATTATGCTGAGTGATATCCCTCT]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC (NNNNNNNN)NNN -5'

5'- NNN(NNNNNNNNN) CTGTCTCTTATACACATCTGACGCTGCCGACGA[TCTCCCTATAGTGAGTCGTATTAATT]CCATG -3'
3'- NNNNNNNNNNNNNN GACAGAGAATATGTGTAGACTGCGACGGCTGCT[AGAGGGATATCACTCAGCATAATTAA]GGTAC -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’- GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNNN -3'



After single strand cDNA synthesis to form cDNA/RNA hybrid

RNA  5’- GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' 
cDNA 3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -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’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'                 (Tm=70, anneal@64C) (P-sss_nXTv2_prmr)
cDNA       3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'


Now have double stranded DNA, which should be long.

DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3' (Tm=70, anneal@64C) (P-sss_nXTv2_prmr)
cDNA       3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN... -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’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCT -3'
cDNA       3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'



After fill in:

DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
cDNA       3'-  CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN 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]GGGAGATCGTCGGCAGC -3'    (i5XX_T7-1_shrt, comp region Tm=60C, whole=76C)
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTC -3' (i5XX_T7-1_long, comp region Tm=65C, whole=77C)
      5’- AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTC -3' (i5XX_orig, comp region Tm=56C, whole=75C)
                                     XXXXXX
                     DNA  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
                     cDNA        3'- CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'
                                                            (i7XX_orig, comp region Tm=57C, whole=73C)   3'- GGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                            (i7XX_long, comp region Tm=59C, whole=73C) 3'- GAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                            (i7_PCR_ILAdptr, Tm=58C)                                           3'- AGCATACGGCAGAAGACGAAC -5'

Read Primers:
Using i5_shrt or i5_long and i7_long
 

         nXT i5 index read ----->                           nXT read1 ----->                        nXT i7 index read ----->
5'- AATGATACGGCGACCACCGAGATCTACAC -3'  5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'      5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3'
5'- AATGATACGGCGACCACCGAGATCTACAC[i5]GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[i7]ATCTCGTATGCCGTCTTCTGCTTG -3'
3'- TTACTATGCCGCTGGTGGCTCTAGATGTG[i5]CCCTCTAGCAGCCGTCGCAGTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[i7]TAGAGCATACGGCAGAAGACGAAC -5'
                                                                                      3'- GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG -5'  
                                                                                          <----- read2 nXT
primers to order:


(T7-nXTv2_i5-1-top) 5'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’
(T7-nXTv2_i5-2-top) 5'- CATGGAATTAATACGACTCACTATAGGGAGACTCCTTCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’
(P-T7tspn-bot)      5'- /5Phos/CTGTCTCTTATACACATCT -3'
(P-sss_nXTv2_prmr)  5’- /5Phos/GGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3'
(nXTv2_i7_top)      5’- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -3'        
(P-nXTv2_i7_bot)    5'- /5Phos/CTGTCTCTTATACACATCT -3'
(i5_PCR_ILAdptr)    5'- AATGATACGGCGACCACCGA -3'
(i7_PCR_ILAdptr)    5'- CAAGCAGAAGACGGCATACGA -3'
(i502_T7-1_shrt)    5'- AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCGTCGGCAGC -3'
(i503_T7-1_shrt)    5'- AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCGTCGGCAGC -3'
(i505_T7-1_shrt)    5'- AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCGTCGGCAGC -3'
(i506_T7-1_shrt)    5'- AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCGTCGGCAGC -3'
(i502_T7-1_long)    5'- AATGATACGGCGACCACCGAGATCTACACCTCTCTATGGGAGATCGTCGGCAGCGTC -3'
(i503_T7-1_long)    5'- AATGATACGGCGACCACCGAGATCTACACTATCCTCTGGGAGATCGTCGGCAGCGTC -3'
(i505_T7-1_long)    5'- AATGATACGGCGACCACCGAGATCTACACGTAAGGAGGGGAGATCGTCGGCAGCGTC -3'
(i506_T7-1_long)    5'- AATGATACGGCGACCACCGAGATCTACACACTGCATAGGGAGATCGTCGGCAGCGTC -3'
(i701_long)         5'- CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGGAG -3'
(i702_long)         5'- CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGGAG -3'
(i703_long)         5'- CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGGAG -3'
(i704_long)         5'- CAAGCAGAAGACGGCATACGAGATGCTCAGGAGTCTCGTGGGCTCGGAG -3'


original Nextera XT V2 i5/i7 barcode sequencers and primers

num.	i5 barcode	i5 name	i5 smplsheet	i5 full sequence
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
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



UPDATE 8/27/2015


Validation of THS-seq update to Nextera XT V2 sequences

  • received tn5059 transposomes T7-nXTv2_i5-1 and nXTv2_i7 from Trina, will run through protocol and MiSeq testing for validation


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
Transposase is undiluted. 1X concentrated
1. idx49, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1
2. idx50, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2
3. idx51, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA
4. idx52, T7-nXTv2_i5-1, 5X tn5-059, NTC



IVT Protocol

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

Generation of sc1-T7iBR-IdxXX transposomes

1. annealing of ME sequence to T7 transposon sequence

    • a. Make 100 uM stock solution of sc1-T7iBR-IdxXX and sc1-T7tspn-NoP-bot.
    • b. Incubate 2.5 uL of each oligo (100uM) with 20 uL EB buffer at 95C for 1 minutes. Oligo's now at 10 uM in 25 uL.
    • c. cool to RT at 0.1 C/s


2. transposome complex generation, run controls!!!

  • add the below components into one tube and incubate for 20 minutes at RT
1. Add 0.5 uL of 100% sterile glycerol to tube
2. Add 0.5 uL of annealed transposon to the 100% sterile glycerol and mix well
4. Add 1.0 uL of DILUTED Ez-TN5 transposase to well.
  • store at -20, is good for a year


Generation of custom barcode (3' side of fragments) nextera transposome

1. annealing of ME sequence to Nextera transposon sequence

    • a. Make 100 uM stock solution of (NXTA_ME_BOT, "pMENTS") and (NXTA_ME_B, "B-METS").
    • b. Incubate 5 uL of each oligo (100uM) with 40 uL EB buffer at 95C for 2 minutes. Oligo's now at 10 uM in 50 uL.
    • c. cool to RT at 0.1 C/s


2. transposome complex generation, run controls!!!

  • add the below components into one tube and incubate for 20 minutes at RT
1.25 uL of annealed Nextera transposon
1.25 uL of 100% sterile glycerol
2.50 uL of Ez-TN5 transposase
  • store at -20, is good for a year


3. Prepare samples, lyse cells with lysis buffer

CELL WASHING - (wash cells with PBS)

  • 1. count cells, spin down all cells at 250-500 g for 5 minutes
  • 2. wash with 1X PBS, centrifuge 250-500 g for 5 minutes, resuspend to XX cells/uL
  • 3. Count cells again, aliquot cells to sample tubes

CELL LYSIS

  • 1. dilute Lysis Buffer (LB) to 2X, or as needed (from 10X stock)
  • 3. add LB to cell aliquots, mix briefly (mineral oil optional)
  • 4. 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, T7-top2)
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.

    • If using beads next add 1.8X (54 uL) beads per sample


ELUTE IN 9.6, leave beads in solution


6. Fill in reaction

  • Add 2.4 uL of 5X taq polymerase or 3.0 uL of taq2X. Run at 72C for 3 minutes. (same as nextera)


8. 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!!!!
  bring to 10 uL with Nuclease free water
  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


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



9. Clean with Zymo clean and concentrator

  • elute samples in 10 uL of N-H2O
  • quanitate with Qubit or on TBU gel.



AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO


Samples this time:

  • sample list
1. idx60, t7-top2, 5X tn5-059, 500 cells rep1
2. idx61, t7-top2, 5X tn5-059, 500 cells rep2
3. idx62, t7-top2, 5X tn5-059, 6 ng pure DNA
4. idx63, t7-top2, 5X tn5-059, NTC
5. idx64, 6/1/2015 6. t7-top2, 5X tn5-059, 1000 cells, N2
6. idx65, 6/1/2015 7. t7-top2, 5X tn5-059, 6 ng pure DNA


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


8. single strand synthesis MMLV RT (Clontech)

20 uL reaction

1. 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

2. heat the mixture to 70C fo 3 minutes.  Immediately cool on ice.

3. 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

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

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



9. 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.


10. Second strand synthesis: Adding 5phos-sss_NPA_prmr primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since 5phos-sss_NPA_prmr has a high Tm.

a. Add 2.5 uL of 5phos-sss_NPA_prmr to each reaction. 

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


d. Add polymerases etc to reactions

          taq2X: Add  5.875 uL, Incubate at 72C for 8 minutes
          
            NTC: Just add 1 uL of Klenow exo-


11. Clean DNA with AMPURE beads depending. elute in 20 uL and concentreate. (can also use qiaquick)

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

can quantitate with Qubit or on TBU gel.


11.5. Eluted DNA into 20 uL per sample, since when eluting in smaller amounts more sample is lost. Thus after elution used vaccum 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. Additionally less transposome will be needed when doing that.

RAN FOR 16 MINUTES


12. Fragmenting and 3' End tagging with Custom Nextera transposome USED Tn5059 AND BUFFER D6, nxta_ME_B

1 uL custom tagmentation buffer
2 uL lysed/pure genomic DNA (X ng/pg DNA)
X uL Nuclase free H2O
1 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) (if was proportional to shendure would use .625 uL) 
___________
5 uL total solution

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


13. 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


14. Fill in reaction

  • Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)


15. PCR addition of barcodes MAKE SURE TO USE NEXTERA INDEXES!!!!!

Samples:


KAPA SYBR FAST qPCR mix until saturation, X35 cycles

9  uL Taq2X
2  uL primers, 2 uL F, (T7-top2-PCR-iaf, OR iaf2, iaf3) USED iaf2
2  uL NXTA indexes PCR_R.NXTAInd[XX], SPECIFIC TO EACH SAMPLE
4  uL H2O
1  uL 25X SYBR green
12 uL DNA template (use half RT reaction)
_____________
30 uL

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

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


16. Gel Size selection

  • gel size select from 400-800 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, 220-800 ish bp


  • gel size selection validation



conclusions

  • submitted for sequencing