Brandon:LabNotes/Project1/2015-8-3: Difference between revisions
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14. Fragmenting and 3' End tagging with custom nXTv2_i7 Tn5059 transposome and custom tagmentation buffer D6 | 14. Fragmenting and 3' End tagging with [1X] custom nXTv2_i7 Tn5059 transposome and custom tagmentation buffer D6 | ||
1 uL custom tagmentation buffer | 1 uL custom tagmentation buffer | ||
X uL 2X cDNA sample | X uL 2X cDNA sample | ||
X uL Nuclase free H2O | X uL Nuclase free H2O | ||
1 uL prepared T7 transposomes (MAKE SURE TO ADD LAST) | 1 uL prepared 1X T7 transposomes (MAKE SURE TO ADD LAST) | ||
___________ | ___________ | ||
5 uL total solution | 5 uL total solution |
Revision as of 03:32, 22 September 2015
Update of THS-seq protocol sequences to use Nextera XT V2 sequences
- THS-seq libraries generated with Testing new method of RNA processing, No RNase III, No polyA polymerase (PAP) protocol have not been working on the MiSeq. Cluster density is really low, and when spiked in is 10-20X less then the total number of reads expected to be generated.
- PCR curves go up as expected, gels, gels size selection, RNA quants are all as expected. Tried replacing all reagents for RNA processing. Tried fresh GM12878 samples. Nothing got it to cluster on MiSeq.
- 2015-6-3 THS miseq troubleshooting R1
- 2015-6-10 THS miseq troubleshooting R2
- 2015-6-18 THS miseq troubleshooting R3
- 2015-7-1 THS miseq troubleshooting R4
- 2015-7-14 THS miseq troubleshooting R5
- 2015-7-24 THS miseq troubleshooting, HiSeq (4/1/2015) Samples that worked, didn't work on MiSeq
- However, these libraries worked on HiSeq, and 2015-04-01 did not work on miseq
- 2015-3-23 Guanidine HCl after transposition, taq2X and IVT on beads, 100 cell samples
- 2015-4-1 Tn5-059 versus ez-tn5 with THS-seq, using 500 cells and guanidine HCl method
- 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.
- Thus will redesign THS-seq using Nextera XT V2 sequencing/index/flowcell/amplification primers.
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 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 T7R1-NEXTERA XT V2 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 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'- CATGGAATTAATACGACTCACTATAGGGAGATCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3’ (T7-nXTv2-1-top) 3'- TCTACACATATTCTCTGTC/5Phos/-5' (P-T7tspn-bot) 5'- /5Phos/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 NNNNNNNNNNNNNN -3' 3'- GTACC[TTAATTATGCTGAGTGATATCCCTCT]AGCAGCCGTCGCAGTCTACACATATTCTCTGTC (NNNNNNNNN)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' 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 NNN--------- 5P/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' PCR barcode addition primers: Using i5_shrt or i5_long and i7_orig or i7_long, test to validate Final sequecing ready fragment nXTv2_i5_index_read -----> nXTv2_read1 -----> nXTv2_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' <----- nXTv2_read2
sequences 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' sequencing primers used: nXTv2_i5_index 5'- AATGATACGGCGACCACCGAGATCTACAC -3' nXTv2_i7_index 5'- CTGTCTCTTATACACATCTCCGAGCCCACGAGAC -3' nXTv2_read1 5'- TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG -3' nXTv2_read2 5'- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG -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
8/27/2015 T7-nXTv2_i5-1 testing
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 1. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1 2. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2 3. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 4. i5XX-i7XX, 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 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 20 minutes at RT
a. Add 0.5 uL of 100% sterile glycerol to tube b. Add 0.5 uL of annealed transposon to the 100% sterile glycerol and mix well c. Add 1.0 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, T7-nXTv2_i5-1) 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 with Qubit or on TBU gel.
AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH ZYMO
sample list 21. i502_shrt-i701_long, 8/27/2015, 1. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1 22. i502_shrt-i702_long, 8/27/2015, 2. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2 23. i503_shrt-i701_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 24. i503_shrt-i702_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 25. i505_shrt-i701_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 26. i505_shrt-i702_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 27. i506_long-i701_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 28. i506_long-i702_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 29. i502_long-i703_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 30. i502_long-i704_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 31. i503_shrt-i703_long, 8/27/2015, 4. T7-nXTv2_i5-1, 5X tn5-059, NTC
- Amounts of RNA and water to add for MMLV RT (50 ng RNA)
9. single strand synthesis MMLV RT (Clontech)
- Followed protocol for SMART MMLV Reverse Transcriptase
20 uL reaction 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 fo 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 P-sss_nXTv2_prmr primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since P-sss_nXTv2_prmr has a high Tm.
a. Add 2.5 uL of 20 uM P-sss_nXTv2_prmr 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 concentreate.
- 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 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.
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 2X 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 i5 AND i7 SEQUENCES!!!!!
Samples: samples processed during RNA processing for rest ofjohn chang T cell samples, first cell division, day7 Tsle and Tmp, day 90 memory, AND nXTv2 THS-seq update RNA processing 21. i502_shrt-i701_long, 8/27/2015, 1. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1 22. i502_shrt-i702_long, 8/27/2015, 2. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2 23. i503_shrt-i701_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 24. i503_shrt-i702_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 25. i505_shrt-i701_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 26. i505_shrt-i702_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 27. i506_long-i701_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 28. i506_long-i702_orig, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 29. i502_long-i703_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 30. i502_long-i704_long, 8/27/2015, 3. T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA 31. i503_shrt-i703_long, 8/27/2015, 4. T7-nXTv2_i5-1, 5X tn5-059, NTC
KAPA SYBR FAST qPCR mix until saturation, X35 cycles 9 uL Taq2X 2 uL nXTv2_i5 index primer, 2 uL F, (i5XX_T7-1_shrt, OR long) 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, 59C for 30s, 72C for 1:30 min) X15, 72C for 3 min, 4C forever 55C for comp ends for taq2X HIGH BACKGROUND WITH 59C, NEXT TIME RUN AT 63C
- 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.
' | 5406.090161 | 5 | ' |
3730.570111 | 2.5 | in 10 uL | |
1. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1 | 17614.20052 | 23.21540477 | 232.1540477 |
2. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2 | 16680.7305 | 21.82259836 | 218.2259836 |
3. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, 6 ng pure DNA | 142250.0542 | 209.1813288 | 2091.813288 |
4. i5XX-i7XX, T7-nXTv2_i5-1, 5X tn5-059, NTC | 708.0800211 | -2.009779054 | -20.09779054 |
- TBU gel after IVT, Guanidine HCl to remove proteins
- yields from 500 cells approx. half that seen before, will try PAGE purified transposon instead, and v2 version.
File:ZhangLab 2 2015-08-28 11hr 27min-labeled.jpg
- qPCR curves
- do not gain much info from efficiency based on PCR curves for short versus long primers. seems 502 works better.
File:2015-09-09 nXTv2 validation, primers testing.bmp
- after adding barcodes, TBE gel
- high amount of primer dimers, maybe since ran at 59C for annealing instead of 63C. next time will try 63C.
File:ZhangLab 2 2015-09-09 18hr 14min-labeled.jpg
- gel size selection amounts
' | 6007.810179 | 2.5 | for 150 ng | X2 |
21. i502_shrt-i701_long, 8/27/2015, 1. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep1 | 23864.16071 | 23.11343865 | 6.48973103 | 12.97946206 |
22. i502_shrt-i702_long, 8/27/2015, 2. T7-nXTv2_i5-1, 5X tn5-059, 500 cells rep2 | 20633.37061 | 19.38380233 | 7.738419813 | 15.47683963 |
- gel size selection
File:ZhangLab 2 2015-09-10 14hr 03min-labeled.jpg
- gel size selection validation
File:ZhangLab 2 2015-09-10 18hr 23min-labeled.jpg
conclusions
- submitted for sequencing