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.





  • 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
GCGCCATCAG][AGATGTGTATAAGAGACAG]

sc-ME-bottom
TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC]

sc-T7-R1-IdxXX
[AAATTAATACGACTCACTATAGGGAGA][TTCTCGCCAAGTCGTCCTTACGGCTC][BARCOD][ATCCTCCCTC


Complete sequence of transposome after ligation

  T7 consensus sequence (27)       N2 Index seq (26)           (6)    for ligation/R1 (20)     ME end (19)
5'- [AAATTAATACGACTCACTATAGGGAGA][TTCTCGCCAAGTCGTCCTTACGGCTC][BARCOD][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][TTCTCGCCAAGTCGTCCTTACGGCTC][BARCOD][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'- AAATTAATACGACTCACTATAGGGAGATTCTCGCCAAGTCGTCCTTACGGCTC[BARCOD]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3'
3'- TTTAATTATGCTGAGTGATATCCCTCTAAGAGCGGTTCAGCAGGAATGCCGAG[BARCOD]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5'




After IVT, RNA generated

RNA  5'- GGGAGATTCTCGCCAAGTCGTCCTTACGGCTC[BARCOD]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3'




After single strand cDNA synthesis to form cDNA/RNA hybrid

RNA  5'- GGGAGATTCTCGCCAAGTCGTCCTTACGGCTC[BARCOD]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3'
cDNA 3'- CCCTCTAAGAGCGGTTCAGCAGGAATGCCGAG[BARCOD]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/GGGAGATTCTCGCCAAGTCGTCCTTACGGCTC -3' (sc-SSS-prmr)
cDNA   3'- CCCTCTAAGAGCGGTTCAGCAGGAATGCCGAG[BARCOD]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5'




Now have double stranded DNA, which should be long

DNA /5Phos/GGGAGATTCTCGCCAAGTCGTCCTTACGGCTC[BARCOD]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN -3'
cDNA   3'- CCCTCTAAGAGCGGTTCAGCAGGAATGCCGAG[BARCOD]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")
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/GGGAGATTCTCGCCAAGTCGTCCTTACGGCTC[BARCOD]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCT -3'
cDNA   3'- CCCTCTAAGAGCGGTTCAGCAGGAATGCCGAG[BARCOD]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGCTAGAGCATACGGCAGAAGACGAAC -5'


After fill in:

DNA /5Phos/GGGAGATTCTCGCCAAGTCGTCCTTACGGCTC[BARCOD]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCTCGATCTCGTATGCCGTCTTCTGCTTG -3'
cDNA   3'- CCCTCTAAGAGCGGTTCAGCAGGAATGCCGAG[BARCOD]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGCTAGAGCATACGGCAGAAGACGAAC -5'


Amplification:
       ILA 5' adaptor
5’- [AATGATACGGCGACCACCGA]GGGAGATTCTCGCCAAGTCGTCCT  sc-T7-PCR1 (comp part (NEB Tm=71), all (NEB Tm=82C)

5’- [AATGATACGGCGACCACCGA]GGGAGATTCTCGCCAAGTCGT     sc-T7-PCR2 (comp part (Tm=67), all (Tm=81C))
 
               DNA /5Phos/GGGAGATTCTCGCCAAGTCGTCCTTACGGCTC[BARCOD]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCTCGATCTCGTATGCCGTCTTCTGCTTG -3'
               cDNA   3'- CCCTCTAAGAGCGGTTCAGCAGGAATGCCGAG[BARCOD]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGCTAGAGCATACGGCAGAAGACGAAC -5'
                                                                                                                                          3'- AGCATACGGCAGAAGACGAAC -5' (NXTA_P2)  (NEB Tm=65C)
                                                                                                                                         GCTAGAGCATACGGCAGAAGACGAAC -5  (NXTA_P2_E)  (NEB Tm=70C)