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

From ZhangLabWiki
Jump to navigation Jump to search
>Bsos
(Created page with "==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...")
 
>Bsos
Line 21: Line 21:
===Sequences information===
===Sequences information===


 
===Summary for using with 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, and possibly RNA fragmentation. Addition of PolyA with E. coli PolyA polymerase (Ambion kit).
Can also try adding adapter to 3'OH end with T4 RNA ligase.
All RNA strands will have same 5' end. First G in GGG is the +1 site for the beginning of RNA synthesis for T7.
RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN... -3'
RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'
T7-frag-block-top2
CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGATCTCCC/3InvdT/





Revision as of 00:13, 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

Summary for using with 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, and possibly RNA fragmentation. Addition of PolyA with E. coli PolyA polymerase (Ambion kit). 
Can also try adding adapter to 3'OH end with T4 RNA ligase.
All RNA strands will have same 5' end. First G in GGG is the +1 site for the beginning of RNA synthesis for T7.

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

RNA 5’- GGGAGATCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNN AAAAAAAAAAAAAAAAAAAAAAAA -3'

T7-frag-block-top2 
CTGTCTCTTATACACATCTCTGATGGCGCGAGGGAGGATCTCCC/3InvdT/





Protocol

  • check is have reagents etc


1. generating custom nextera transposome


2.