Brandon:LabNotes/Project1/2013-12-3
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Transposon for single cell accessibility
- 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
sc-ME-top 5'- GCGCCATCAG][AGATGTGTATAAGAGACAG] -3' sc-ME-bottom 3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] -5' sc-T7-R1-IdxXX 5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][XXXXXX][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'- GCGCCATCAG][AGATGTGTATAAGAGACAG] -3' sc-ME-top 3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] -5' sc-ME-bottom After transposition (fragment from two adjacent insertions) 5'- |GCGCCATCAG][AGATGTGTATAAGAGACAG] NNNNNNNNNNN--------- [CTGTCTCTTATACACATCT][CTGATGGCGC|GAGGGAGGAT -3' 3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] ---------NNNNNNNNNNN [GACAGAGAATATGTGTAGA][GACTACCGCG -5' Single insertion 5'- NN--------- [CTGTCTCTTATACACATCT][CTGATGGCGC|GAGGGAGGAT |GCGCCATCAG][AGATGTGTATAAGAGACAG] NNNNNNNNNNN -3' 3'- NNNNNNNNNNN [GACAGAGAATATGTGTAGA][GACTACCGCG 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) 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' (NXTA_ME_BOT, "pMENTS") 3'- GACAGAGAATATGTGTAGAGC[TAGAGCATACGGCAGAAGACGAAC] -5' (sc-ILA-adaptor) 3' ILA adaptor 3'- TCTACACATATTCTCTGTC/5Phos/ (NXTA_ME_BOT, "pMENTS") 3'- [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-PCR1 (comp part (NEB Tm=72), all (NEB Tm=83C) 5’- [AATGATACGGCGACCACCGA]GGGAGAAAGAGCGGTTCAGCA sc-T7-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' (NXTA_P2) (NEB Tm=65C) 3'- GC[TAGAGCATACGGCAGAAGACGAAC -5 (NXTA_P2_E) (NEB Tm=70C) Read primers (N2 Index read) T7tspn-Read1 (Tm=75.80) 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'