Brandon:LabNotes/Project1/2013-12-3
Transposon for single cell accessibility (T4 ligation)[edit]
- Know that transposon works well for 1000 cells and produces alot of RNA product after IVT amplification. However there are limitations to amplification by IVT, as ~400 pg of DNA is needed for amplification by IVT. To try and overcome this limitation, single cells can be barcoded upon tranposition, then pooled for IVT amplification.
- no cleaning steps required
- dilute transposome, found can still work well with 8X dilution. (still waiting for sequencing results on other dilution. However this is for 500 cells.
- There are two papers examining single cell transcriptomics with relevant methods.
- Quartz-Seq: a highly reproducible and sensitive single-cell RNA sequencing method, reveals non-genetic gene-expression heterogeneity
- use IVT for labeling, but PCR for amplification of cDNA. Single cells are isolated with FACS into a plate with .4 uL of lysis buffer that has a bubble, to ensure the cell is captured in the solution.
- CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification
- pools samples are performing reverse transcription and second strand synthesis to create a DNA template for T7. ~400 pg of total sample is needed for a viable T7 amplification. Will pool enough cells to obtain this amount of template.
- Tested IVT amplification on 50 cells and found enough RNA (~50 ng is generated to perform the barcoding reaction). however since barcoding is already done, downstream processing will be different.
- Thus 50-100 cells pooled after barcoding looks feasible.
- Possible issues
- loss of product when pooling samples. more efficient way to pool?
- Need to use DNA lo-bind tubes
- T4 ligation efficiency?
Single cell transposon design[edit]
sc-ME-top 5'- /5Phos/GCGCCATCAG][AGATGTGTATAAGAGACAG] -3' sc-ME-bottom 3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC]/5Phos/ -5' 5'- /5Phos/[CTGTCTCTTATACACATCT]CTGATGGCGC|GAGGGAGGAT -3' sc-T7-R1-IdxXX 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][XXXXXX][ATCCTCCCTC] -3' sc-T7-R1-Idx49 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][CTGTGT][ATCCTCCCTC] -3' sc-T7-R1-Idx50 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][ACCTTT][ATCCTCCCTC] -3' sc-T7-R1-Idx51 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][TATCGC][ATCCTCCCTC] -3' sc-T7-R1-Idx52 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][GACACG][ATCCTCCCTC] -3' sc-T7-R1-Idx53 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][TTCTAC][ATCCTCCCTC] -3' sc-T7-R1-Idx54 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][ACGTCC][ATCCTCCCTC] -3' sc-T7-R1-Idx55 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][TCGACT][ATCCTCCCTC] -3' sc-T7-R1-Idx56 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][TCAGAC][ATCCTCCCTC] -3' sc-T7-R1-Idx57 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][TCCTTC][ATCCTCCCTC] -3' sc-T7-R1-Idx58 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][CAGCAT][ATCCTCCCTC] -3' sc-T7-R1-Idx59 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][GATAGA][ATCCTCCCTC] -3' sc-T7-R1-Idx60 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][ACAGAT][ATCCTCCCTC] -3' sc-T7-R1-Idx61 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][CTCTAT][ATCCTCCCTC] -3' Complete sequence of transposome after ligation T7 consensus sequence (27) N2 Index seq (26) bc(6) for ligation/R1 (20) ME end (19) 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][XXXXXX][ATCCTCCCTC|GCGCCATCAG][AGATGTGTATAAGAGACAG] -3' After annealing sc-ME-annealed 5'- /5Phos/GCGCCATCAG][AGATGTGTATAAGAGACAG] -3' sc-ME-top 3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC]/5Phos/ -5' sc-ME-bottom After transposition (fragment from two adjacent insertions) 5'- /5Phos/|GCGCCATCAG][AGATGTGTATAAGAGACAG] NNNNNNNNNNN--------- [CTGTCTCTTATACACATCT][CTGATGGCGC|GAGGGAGGAT -3' 3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] ---------NNNNNNNNNNN [GACAGAGAATATGTGTAGA][GACTACCGCG/5Phos/ -5' Single insertion 5'- NN--------- [CTGTCTCTTATACACATCT][CTGATGGCGC|GAGGGAGGAT /5Phos/|GCGCCATCAG][AGATGTGTATAAGAGACAG] NNNNNNNNNNN -3' 3'- NNNNNNNNNNN [GACAGAGAATATGTGTAGA][GACTACCGCG/5Phos/ TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] ---------NN -5' After ligation with T4 (amp ligase?) (single insertion) 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][XXXXXX][ATCCTCCCTC|GCGCCATCAG][AGATGTGTATAAGAGACAG] NNNNNNNNNNN -3' sc-ME-top + sc-T7-R1-Idx49 3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] ---------NN -5' sc-ME-bottom After gap fill in/extension with taq2x 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' 3'- TTTAATTATGCTGAGTGATATCCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5' After IVT, RNA generated RNA 5'- GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' After single strand cDNA synthesis to form cDNA/RNA hybrid RNA 5'- GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -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 DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG -3' (sc-SSS-prmr)(NEB Tm=78C) cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5' Now have double stranded DNA, which should be long DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5'
Normal Nextera 5' end: 5'-GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG-3' ("A-METS" NXTA_ME_A) 3'- TCTACACATATTCTCTGTC/5Phos/ (NXTA_ME_BOT, "pMENTS") after fill in: 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' 3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5' PCR amplification: 5'-AATGATACGGCGACCACCGA-3' (NXTA_P1) 5'-AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAG-3' (NXTA_adpt1) 5'- GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3' 3'- CGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5'
Custom Nextera transposome for 3' end 5'- /5Phos/CTGTCTCTTATACACATCT -3' (sc-NXTA_ME_bot, "pMENTS") 3'- GACAGAGAATATGTGTAGAGC[TAGAGCATACGGCAGAAGACGAAC] -5' (sc-ILA-adaptor) 3' ILA adaptor 3'- TCTACACATATTCTCTGTC/5Phos/ (sc-NXTA_ME_bot, "pMENTS") 5'- [CAAGCAGAAGACGGCATACGAGAT]CGAGATGTGTATAAGAGACAG -3' (sc-ILA-adaptor) 3' ILA adaptor After custom nextera tagmentation: Now have double stranded DNA, which should be long DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCT -3' cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGCTAGAGCATACGGCAGAAGACGAAC -5' After fill in: DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCTCGATCTCGTATGCCGTCTTCTGCTTG -3' cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGCTAGAGCATACGGCAGAAGACGAAC -5' Amplification: ILA 5' adaptor 5’- [AATGATACGGCGACCACCGA]GGGAGAAAGAGCGGTTCAGCAGGA sc-T7-5'-PCR1 (comp part (NEB Tm=72), all (NEB Tm=83C) 5’- [AATGATACGGCGACCACCGA]GGGAGAAAGAGCGGTTCAGCA sc-T7-5'-PCR2 (comp part (Tm=68), all (Tm=81C)) ILA 3' adaptor DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCTCG[ATCTCGTATGCCGTCTTCTGCTTG] -3' cDNA 3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGC[TAGAGCATACGGCAGAAGACGAAC] -5' 3'- AGCATACGGCAGAAGACGAAC -5' (sc-NXTA_3'-P2) (NEB Tm=65C) 3'- GC[TAGAGCATACGGCAGAAGACGAAC -5 (sc-NXTA_P2-3'_E) (NEB Tm=70C) Read primers (N2 Index read) T7tspn-Read1 (Tm=75.80) (same as NXTA R1) ILA 5' adaptor AAGAGCGGTTCAGCAGGAATGCCGAG TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG ILA 3' adaptor 5’- [AATGATACGGCGACCACCGA]GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCTCG[ATCTCGTATGCCGTCTTCTGCTTG] -3' 3'- [TTACTATGCCGCTGGTGGCT]CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGC[TAGAGCATACGGCAGAAGACGAAC] -5'
primers ordered sc-ME-top 5'- /5Phos/GCGCCATCAGAGATGTGTATAAGAGACAG -3' sc-ME-bottom 5'- /5Phos/CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGAT -3' sc-SSS-prmr /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG -3' sc-ILA-adaptor CAAGCAGAAGACGGCATACGAGATCGAGATGTGTATAAGAGACAG sc-NXTA_ME_bot /5Phos/CTGTCTCTTATACACATCT sc-T7-5'-PCR1 AATGATACGGCGACCACCGAGGGAGAAAGAGCGGTTCAGCAGGA sc-T7-5'-PCR2 AATGATACGGCGACCACCGAGGGAGAAAGAGCGGTTCAGCA sc-NXTA-3'_P2 CAAGCAGAAGACGGCATACGA sc-NXTA-3'_P2_E CAAGCAGAAGACGGCATACGAGATCG sc-T7-R1-Idx49 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGCTGTGTATCCTCCCTC -3' sc-T7-R1-Idx50 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGACCTTTATCCTCCCTC -3' sc-T7-R1-Idx51 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGTATCGCATCCTCCCTC -3' sc-T7-R1-Idx52 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGGACACGATCCTCCCTC -3' sc-T7-R1-Idx53 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGTTCTACATCCTCCCTC -3' sc-T7-R1-Idx54 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGACGTCCATCCTCCCTC -3' sc-T7-R1-Idx55 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGTCGACTATCCTCCCTC -3' sc-T7-R1-Idx56 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGTCAGACATCCTCCCTC -3' sc-T7-R1-Idx57 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGTCCTTCATCCTCCCTC -3' sc-T7-R1-Idx58 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGCAGCATATCCTCCCTC -3' sc-T7-R1-Idx59 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGGATAGAATCCTCCCTC -3' sc-T7-R1-Idx60 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGACAGATATCCTCCCTC -3' sc-T7-R1-Idx61 5'- AAATTAATACGACTCACTATAGGGAGAAAGAGCGGTTCAGCAGGAATGCCGAGCTCTATATCCTCCCTC -3'
Protocol[edit]
IVT Protocol[edit]
- If need to make more transposome, do first 2 steps. If not goto step 3.
Generation of sc transposome
1. annealing of ME sequence top and bottom
- a. Make 100 uM stock solution of sc-ME-top and sc-ME-bottom.
- 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 transposon 1.25 uL of 100% sterile glycerol 2.50 uL of Ez-TN5 transposase
- store at -20, is good for a year
Generation of 3' ILA adaptor transposome
1. annealing of ME sequence to Nextera transposon sequence
- a. Make 100 uM stock solution of (NXTA_ME_BOT, "pMENTS") and (sc-ILA-adaptor).
- 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
sample list 1. XX 2. XX
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 XXXX cells/uL
- 3. Aliquot cells to sample tubes
CELL LYSIS
- 1. dilute LB to 2X, or as needed (from 10X stock)
- 2. refrigerate or warm LB or keep at RT
- 3. add lysis buffer 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, using (sc transposon)
SORT SAMPLES WITH FACS, MAKE 5 uL BUBBLE 1-2 uL OF LYSIS BUFFER IN 200 uL TUBES
- add the below into one tube and incubate for 10 minutes at 55C.
.5 uL nextera LMW buffer 2 uL lysed/pure genomic DNA (X ng/pg DNA) 0 uL Nuclease free water 1 uL prepared and diluted T7 transposomes (MAKE SURE TO ADD LAST) OR DILUTED TSPN, 4X, 8X, 16X ___________ 3.5 uL total solution
5. Protease digestion of transposase, protease inactivation
To each tube, add: .5 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
6. T4 ligation of T7 promoter and barcodes.
.5 uL add sc-T7-R1-IdxXX (10 uM) to each sample .5 uL of T4 DNA ligase. .5 uL T4 buffer (10X?) Incubate at 20C for 10 minutes Inactivate 65C for 10 minutes
7. Fill in reaction
- Add 5 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)
8. Pool samples and clean with standard EtOH precipitation.
- resuspend in 6 uL N-H2O
9. Maxiscript (Ambion) T7 Protocol, IVT
CAN TRY IN 20 uL REACTION TOO
- 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) 1 uL 10X Transcription Buffer .5 uL 10 mM ATP .5 uL 10 mM CTP .5 uL 10 mM GTP .5 uL 10 mM UTP 1 uL T7 Enzyme Mix b. Incubate reactions at 37C overnight for ~16 hours. (>10 uM limiting nucleotide)
CLEAN WITH ZYMO RNA CLEAN AND CONCENTRATOR NOW
10. Clean RNA with AMPURE RNA beads. elute in 20 uL.
- Protocol changes:
- 1. add 30 uL to IVT reaction to bring to 50 uL total before starting protocol.
- 2. Use 1.8:1 ratio of beads, thus 80 uL of beads for 50 uL of sample in this case
- 3. Drying time after the washes is 3 minutes.
- elute in 20 uL and concentrate to 10 uL
- can quantitate with Qubit or on TBU gel.
AFTER IVT RNA HAS ALREADY BEEN GENERATED AND CLEANED WITH AMPURE BEADS
Samples this time:
sample list 1. XX
- Amounts of RNA and water to add for MMLV RT (50 ng RNA)
11. single strand synthesis MMLV RT (Clontech)
- Followed protocol for SMART MMLV Reverse Transcriptase
20 uL reaction 1. Add 2.5 uL 20 uM (100 ng total) RANDOM HEXAMERS to RNA sample. Bring to final volume of 12.5 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. 2 uL 5X first strand buffer 2 uL dNTP mix 2 uL 100 mM DTT 1 uL N-H20 .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
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 sc-SSS-prmr primer and polymerases. Can incubate at higher temps (usually at 16C since RNA is nicked) since sss_NPA_prmr has a high Tm.
a. Add 2.5 uL of sc-SSS-prmr to each reaction. b. Incubate solutions for 2 minutes at 65C. Cool immediately on ice. c. Add 2 uL of dNTPs to each reaction. d. Add polymerases etc to reactions can also use DNAP1, klenow exo-, BST 2.0, or BST 2.0 without buffer. for more information see second strand synthesis section taq5X: Add 5 uL, Incubate at 72C for 10 minutes
14. Clean DNA with with Zymo DNA Clean & Concentrator-5 Kit OR AMPURE beads depending. elute in 20 uL and concentrate . (can also use qiaquick)
NEED TO USE AMPURE DNA BEADS AT 1:1.8X RATIO
can quantitate with Qubit or on TBU gel.
14.5. 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.
15. Fragmenting and 3' End tagging with Custom Nextera transposome
1 uL nextera LMW buffer 2 uL lysed/pure genomic DNA (X ng/pg DNA) X uL Nuclase free H2O .4 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 10 minutes, cool briefly on ice after
16. 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
17. Fill in reaction
- Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)
18. PCR amplification
Samples: 1. XX
KAPA SYBR FAST qPCR mix until saturation, X35 cycles 9 uL Taq2X 2 uL F primers, 2 uL (sc-T7-PCR1, OR sc-T7-PCR2) 2 uL R primer , 2 uL (NXTA_P2 or NXTA_PE_E) 4 uL H2O 1 uL 25X SYBR green 12 uL DNA template (use half RT reaction) _____________ 30 uL KAPA SYBR cycles: 95C 3min, (95C for 30s, 60C for 30s, 72C for 2 min) X35, 72C for 5 min, 4C forever
- terminate before curves saturate (usually cycle 6-8)
19. Gel Size selection
- gel size select from 400-800 bp, follow gel size selection protocol
- do not need to include controls.