Brandon:LabNotes/Project1/2013-7-10: Difference between revisions

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>Bsos
>Bsos
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===Sequences information===
===Sequences information, Using T7tspn-top2===
 
===Summary for using with T7tspn-top2:===
   
   
  Sequences:
  Sequences:
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  After IVT, and possibly RNA fragmentation. Addition of PolyA with E. coli PolyA polymerase (Ambion kit).
  After IVT, All RNA strands will have same 5' end.  
Can also try adding adapter to 3'OH end with T4 RNA ligase.
First G in GGG is the +1 site for the beginning of RNA synthesis for T7.
All RNA strands will have same 5' end. 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)
   
   
  RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'
  RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'
   
   
RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'
   
   
T7-frag-block-top2
CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGATCTCCC/3InvdT/
   
   
After single strand cDNA synthesis to form cDNA/RNA hybrid
   
   
RNA  5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'
cDNA 3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'
   
   
   
   
After Rnase H nicking, denatuation of small RNAs? and addition of 5' end primer.
DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'(Tm=79) (sss_NPA_prmr) (No Poly A)
cDNA  3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'
Now have double stranded DNA, which should be long.
DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'
cDNA  3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN... -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
5'- /5Phos/CTGTCTCTTATACACATCT -3' (NXTA_ME_BOT, "pMENTS")
        3'- GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'  (NXTA_ME_B, "B-METS")
                          3'- TCTACACATATTCTCTGTC/5Phos/ (NXTA_ME_BOT, "pMENTS")
        5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG -3'  (NXTA_ME_B, "B-METS")
After custom nextera tagmentation:
DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCT -3'
cDNA  3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
After fill in:
DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
cDNA  3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
Amplification:
5’- [AATGATACGGCGACCACCGA]GGG]AGATCCTCCCTCGCGCCATCAGAG    T7-top2-PCR-iaf3 (comp part (Tm=77), all (Tm=85C)
                 
    5’- [AATGATACGGCGACCACCGA]AGATCCTCCCTCGCGCCATCAGAG    T7-top2-PCR-iaf2 (comp part (Tm=73), all (Tm=83C))
                          X X
    5’- [AATGATACGGCGACCACCGA]GATCTCTCCCTCGCGCCATCAGAGAT  T7-top2-PCR-iaf (original) (comp region 70C) (whole Tm=83C)
                          X X XXXXX
                DNA /5Phos/GGG]AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
                cDNA  3'- CCC]TCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
                                                                                                    3'- GACTCGCCCGACCGTTCCGTCTGGC[GACACA]TAGAGCATACGGCAGAAGACGAAC -5' (custom barcodes) (Tm=86C)
                                                                                                    3'- GACTCGCCCGACCGTTCCGTCTGGC[------]TAGAGCATACGGCAGAAGACGAAC -5' (NXTA_adpt2)(comp region Tm=73C) (whole Tm=85C)
                                                                                                                                        3'- AGCATACGGCAGAAGACGAAC -5' (NXTA_P2)





Revision as of 02:42, 10 July 2013

Testing new method of RNA processing, No RNase III, No polyA polymerase (PAP)

  • In performing the Tn5 accessibility method, the most troubling steps are when RNase III is used for fragmentation and polyA polymerase is used to add polyA tails to the 3' end, so the T20VN_PE_R primer can be used to add adaptors/barcodes etc. Thus this protocol avoids the usage of both enzymes. RNase III does not seem to have a good shelf life and does not function correctly if it is not taken care of, and stops working approximately 3 months after purchase. PAP is a extremely robust enzyme, and adds polyA to most segments. Most of the unmapped reads are polyA tailed fragments with the sequencing reads consisting of polyA sequences.


  • Changes:


    • Another robust method of generating cDNA will be used, using random hexamers for priming and MMLV for RT


    • RNA in the RNA/cDNA hybrid will be degraged with RNase H, and heated for denaturing. Next, extension with BST 2.0 or Klenow exo- will be done with a DNA primer to the 5' end.


    • DNA will will need to be fragmented, but this time a custom transpsome will be created that only has the nextera B ends on it, the 3' end of fragments were barcodes are attached.



Sequences information, Using T7tspn-top2

Sequences:

5'- [CATGAGA][TTAATACGACTCACTATAG][GGAGA][TCCTCCCTCGCGCCATCA][G][AGATGTGTATAAGAGACAG] -3’  (T7tspn-top2)
       spcr      T7 forward    required spcr   read sequence  spcr       ME 

5'- [phos]CTGTCTCTTATACACATCT -3' (T7tspn-bot)

 T7 description:
                                                        +1
                              CATGAGA[TTAATACGACTCACTATA|G|GGAGA]
[end sequence for optimal binding]         T7 -->          [front sequence for optimal binding]



After Annealing:

       5’- CATGAGATTAATACGACTCACTATAGGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3’         (T7tspn-top2)
                                                         3'- TCTACACATATTCTCTGTC [Phos]-5'   (T7tspn-bot)

5'- [phos] CTGTCTCTTATACACATCT -3'
      3’-  GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCTAGAGGGATATCACTCAGCATAATTAGAGTAC -5’


Fragments created after transposition:


5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNN------ CTGTCTCTTATACACATCT -3'
                                                    3'- TCTACACATATTCTCTGTC ------NNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’ 


Upon single insertion, T7 still present in either direction of insertion, IVT off top strand, and bottom:

NNNNN------ CTGTCTCTTATACACATCT -3'              5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNN
NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’              3'- TCTACACATATTCTCTGTC ------NNNN



After fill in with taq polymerase (2X) (could use BST, T4 DNA poly also)

5’- CATGAGA[TTAATACGACTCACTATAGGGAGA]TCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN
3'- GTACTCT[AATTATGCTGAGTGATATCCCTCT]AGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC (NNNNNN)NNNN


NNN(NNNNNN) CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGA[TCTCCCTATAGTGAGTCGTATTAA]TCTCATG -3'
NNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCT[AGAGGGATATCACTCAGCATAATT]AGAGTAC -5’



After IVT, All RNA strands will have same 5' end. 
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)

RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'



After single strand cDNA synthesis to form cDNA/RNA hybrid

RNA  5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'
cDNA 3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'



After Rnase H nicking, denatuation of small RNAs? and addition of 5' end primer.

DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG -3'(Tm=79) (sss_NPA_prmr) (No Poly A)
cDNA   3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN... -5'


Now have double stranded DNA, which should be long.

DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'
cDNA   3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN... -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
5'- /5Phos/CTGTCTCTTATACACATCT -3' (NXTA_ME_BOT, "pMENTS")
       3'- GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'  (NXTA_ME_B, "B-METS")


                          3'- TCTACACATATTCTCTGTC/5Phos/ (NXTA_ME_BOT, "pMENTS")
       5'- GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG -3'  (NXTA_ME_B, "B-METS")


After custom nextera tagmentation:

DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCT -3'
cDNA   3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'



After fill in:

DNA /5Phos/GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
cDNA   3'- CCCTCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'


Amplification:
5’- [AATGATACGGCGACCACCGA]GGG]AGATCCTCCCTCGCGCCATCAGAG     T7-top2-PCR-iaf3 (comp part (Tm=77), all (Tm=85C)
                 
    5’- [AATGATACGGCGACCACCGA]AGATCCTCCCTCGCGCCATCAGAG     T7-top2-PCR-iaf2 (comp part (Tm=73), all (Tm=83C))
                          X X 
    5’- [AATGATACGGCGACCACCGA]GATCTCTCCCTCGCGCCATCAGAGAT  T7-top2-PCR-iaf (original) (comp region 70C) (whole Tm=83C) 
                          X X XXXXX
               DNA /5Phos/GGG]AGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGC -3'
               cDNA   3'- CCC]TCTAGGAGGGAGCGCGGTAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG -5'
                                                                                                    3'- GACTCGCCCGACCGTTCCGTCTGGC[GACACA]TAGAGCATACGGCAGAAGACGAAC -5' (custom barcodes) (Tm=86C)
                                                                                                    3'- GACTCGCCCGACCGTTCCGTCTGGC[------]TAGAGCATACGGCAGAAGACGAAC -5' (NXTA_adpt2)(comp region Tm=73C) (whole Tm=85C)
                                                                                                                                        3'- AGCATACGGCAGAAGACGAAC -5' (NXTA_P2)




Protocol

  • check is have reagents etc


1. generating custom nextera transposome


2.