Brandon:LabNotes/Project1/2013-12-3: Difference between revisions

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==Transposon for single cell accessibility==
==Transposon for single cell accessibility (T4 ligation)==




Line 33: Line 33:


  sc-ME-top
  sc-ME-top
  5'- GCGCCATCAG][AGATGTGTATAAGAGACAG] -3'
  5'- /5Phos/GCGCCATCAG][AGATGTGTATAAGAGACAG] -3'
   
   
  sc-ME-bottom
  sc-ME-bottom
  3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] -5'
  3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC]/5Phos/ -5'
5'- /5Phos/[CTGTCTCTTATACACATCT]CTGATGGCGC|GAGGGAGGAT -3'
   
   
  sc-T7-R1-IdxXX                 
  sc-T7-R1-IdxXX                 
  5'- [AAATTAATACGACTCACTATAGGGAGA][AAGAGCGGTTCAGCAGGAATGCCGAG][XXXXXX][ATCCTCCCTC -3'
  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
  Complete sequence of transposome after ligation
Line 50: Line 67:
  After annealing  
  After annealing  
  sc-ME-annealed
  sc-ME-annealed
            5'- GCGCCATCAG][AGATGTGTATAAGAGACAG] -3' sc-ME-top
    5'- /5Phos/GCGCCATCAG][AGATGTGTATAAGAGACAG] -3' sc-ME-top
  3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] -5' sc-ME-bottom
  3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC]/5Phos/ -5' sc-ME-bottom
   
   
   
   
Line 57: Line 74:
  After transposition (fragment from two adjacent insertions)
  After transposition (fragment from two adjacent insertions)
   
   
            5'- |GCGCCATCAG][AGATGTGTATAAGAGACAG] NNNNNNNNNNN--------- [CTGTCTCTTATACACATCT][CTGATGGCGC|GAGGGAGGAT -3'
    5'- /5Phos/|GCGCCATCAG][AGATGTGTATAAGAGACAG] NNNNNNNNNNN--------- [CTGTCTCTTATACACATCT][CTGATGGCGC|GAGGGAGGAT -3'
   3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] ---------NNNNNNNNNNN [GACAGAGAATATGTGTAGA][GACTACCGCG -5'
   3'- TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] ---------NNNNNNNNNNN [GACAGAGAATATGTGTAGA][GACTACCGCG/5Phos/ -5'
   
   
   
   
Line 64: Line 81:
  Single insertion
  Single insertion
   
   
  5'- NN--------- [CTGTCTCTTATACACATCT][CTGATGGCGC|GAGGGAGGAT             |GCGCCATCAG][AGATGTGTATAAGAGACAG] NNNNNNNNNNN -3'
  5'- NN--------- [CTGTCTCTTATACACATCT][CTGATGGCGC|GAGGGAGGAT       /5Phos/|GCGCCATCAG][AGATGTGTATAAGAGACAG] NNNNNNNNNNN -3'
  3'- NNNNNNNNNNN [GACAGAGAATATGTGTAGA][GACTACCGCG               TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] ---------NN -5'
  3'- NNNNNNNNNNN [GACAGAGAATATGTGTAGA][GACTACCGCG/5Phos/        TAGGAGGGAG|CGCGGTAGTC][TCTACACATATTCTCTGTC] ---------NN -5'
   
   
   
   
Line 103: Line 120:
  Extension with BST or klenow exo- or DNAP1 or Taq2x
  Extension with BST or klenow exo- or DNAP1 or Taq2x
   
   
  DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG -3' (sc-SSS-prmr)
  DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG -3' (sc-SSS-prmr)(NEB Tm=78C)
  cDNA  3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5'
  cDNA  3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN -5'
   
   
Line 139: Line 156:
  Custom Nextera transposome for 3' end
  Custom Nextera transposome for 3' end
   
   
  5'- /5Phos/CTGTCTCTTATACACATCT -3' (NXTA_ME_BOT, "pMENTS")
  5'- /5Phos/CTGTCTCTTATACACATCT -3' (sc-NXTA_ME_bot, "pMENTS")
         3'- GACAGAGAATATGTGTAGAGC[TAGAGCATACGGCAGAAGACGAAC] -5'  (sc-ILA-adaptor)
         3'- GACAGAGAATATGTGTAGAGC[TAGAGCATACGGCAGAAGACGAAC] -5'  (sc-ILA-adaptor)
                                       3' ILA adaptor
                                       3' ILA adaptor
   
   
   
   
                             3'- TCTACACATATTCTCTGTC/5Phos/ (NXTA_ME_BOT, "pMENTS")
                             3'- TCTACACATATTCTCTGTC/5Phos/ (sc-NXTA_ME_bot, "pMENTS")
  3'- [CAAGCAGAAGACGGCATACGAGAT]CGAGATGTGTATAAGAGACAG -3'  (sc-ILA-adaptor)
  5'- [CAAGCAGAAGACGGCATACGAGAT]CGAGATGTGTATAAGAGACAG -3'  (sc-ILA-adaptor)
             3' ILA adaptor
             3' ILA adaptor
   
   
Line 166: Line 183:
  Amplification:
  Amplification:
         ILA 5' adaptor
         ILA 5' adaptor
  5’- [AATGATACGGCGACCACCGA]GGGAGAAAGAGCGGTTCAGCAGGA  sc-T7-PCR1 (comp part (NEB Tm=72), all (NEB Tm=83C)
  5’- [AATGATACGGCGACCACCGA]GGGAGAAAGAGCGGTTCAGCAGGA  sc-T7-5'-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
  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'
                 DNA /5Phos/GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCTCG[ATCTCGTATGCCGTCTTCTGCTTG] -3'
                 cDNA  3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGC[TAGAGCATACGGCAGAAGACGAAC] -5'
                 cDNA  3'- CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGC[TAGAGCATACGGCAGAAGACGAAC] -5'
                                                                                                                                             3'- AGCATACGGCAGAAGACGAAC -5' (NXTA_P2)  (NEB Tm=65C)
                                                                                                                                             3'- AGCATACGGCAGAAGACGAAC -5' (sc-NXTA_3'-P2)  (NEB Tm=65C)
                                                                                                                                       3'- GC[TAGAGCATACGGCAGAAGACGAAC -5  (NXTA_P2_E)  (NEB Tm=70C)
                                                                                                                                       3'- GC[TAGAGCATACGGCAGAAGACGAAC -5  (sc-NXTA_P2-3'_E)  (NEB Tm=70C)
   
   
   
   
  Read primers
  Read primers
   
   
                                     (N2 Index read)                     T7tspn-Read1 (Tm=75.80)
                                     (N2 Index read)               T7tspn-Read1 (Tm=75.80) (same as NXTA R1)  
         ILA 5' adaptor          AAGAGCGGTTCAGCAGGAATGCCGAG        TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG                                      ILA 3' adaptor
         ILA 5' adaptor          AAGAGCGGTTCAGCAGGAATGCCGAG        TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG                                      ILA 3' adaptor
  5’- [AATGATACGGCGACCACCGA]GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCTCG[ATCTCGTATGCCGTCTTCTGCTTG] -3'
  5’- [AATGATACGGCGACCACCGA]GGGAGAAAGAGCGGTTCAGCAGGAATGCCGAG[XXXXXX]ATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNN CTGTCTCTTATACACATCTCG[ATCTCGTATGCCGTCTTCTGCTTG] -3'
  3'- [TTACTATGCCGCTGGTGGCT]CCCTCTTTCTCGCCAAGTCGTCCTTACGGCTC[XXXXXX]TAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNN GACAGAGAATATGTGTAGAGC[TAGAGCATACGGCAGAAGACGAAC] -5'
  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'




Line 235: Line 278:




'''CELL SAMPLES (ALL)'''
CELL WASHING - (wash cells with PBS)
*1. refrigerate and warm LB.
*1. count cells, spin down all cells at 250-500 g for 5 minutes
*2. add warm or cold lysis buffer to cell aliquots, mix briefly
*2. wash with 1X PBS, centrifuge 250-500 g for 5 minutes, resuspend to XXXX cells/uL
*3. transposition ready
*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




*Make 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.  
LYSIS BUFFER NOTES
**spin down cells to concentrate them as necessary.
*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.
*Prepare 2X LB from 10X buffer. mineral oil optional.
*if needed make 4 or 6 uL solutions. keep 1:1 ratio of cells:lysis buffer




Line 251: Line 297:
4. transposition reaction, using (sc transposon)
4. transposition reaction, using (sc transposon)


 
'''SORT SAMPLES WITH FACS, MAKE 5 uL BUBBLE 1-2 uL OF LYSIS BUFFER IN 200 uL TUBES'''
'''SAMPLES (ALL), 55c 10 MINUTES'''
*add the below into one tube and incubate for 10 minutes at 55C.
*add the below into one tube and incubate for 10 minutes at 55C.
  .5 uL nextera LMW buffer
  .5 uL nextera LMW buffer
Line 401: Line 446:




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.
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.
*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.


Line 408: Line 453:




12. Fragmenting and 3' End tagging with Custom Nextera transposome
15. Fragmenting and 3' End tagging with Custom Nextera transposome


  1 uL nextera LMW buffer
  1 uL nextera LMW buffer
Line 423: Line 468:




13. Protease digestion of transposase, protease inactivation
16. Protease digestion of transposase, protease inactivation
  To each tube, add:
  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 [])
  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 [])
Line 432: Line 477:




14. Fill in reaction
17. Fill in reaction
*Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)
*Add 6 uL 2X taq polymerase, run at 72C for 3 minutes. (same as nextera)




15. PCR amplification
18. PCR amplification


  Samples:
  Samples:
Line 460: Line 505:




16. Gel Size selection
19. Gel Size selection
*gel size select from 400-800 bp, follow gel size selection protocol
*gel size select from 400-800 bp, follow gel size selection protocol
*do not need to include controls.
*do not need to include controls.

Latest revision as of 01:52, 31 January 2014

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.





  • 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)

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.



results[edit]