Athurva Gore:LabNotes/2012-4-26

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Summary of Primers in Shendure Paper[edit]

  • Paper: Ultra-low-input, tagmentation-based whole genome bisulfite sequencing [[1]]
  • What do we need to replicate this protocol?
    • KAPA Robust PCR amplification kit (have this)
    • AmpLigase (have this)
    • EZ-Tn5 Transposase (ordered more)
    • Primers. See list below:
Transposons:

5' - GATCTACACGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG - 3’
                                3' - CACATATTCTCTGTC - 5'

5' - CTGTCTCTTATACAC - 3'
3' - GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCGCACATCTAG - 5'


After transposition:

5' - GATCTACACGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNN--------- CTGTCTCTTATACAC
                                     CACATATTCTCTGTC ---------NNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCGCACATCTAG - 5'



Adaptor 2 Annealing:
5' - CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCGA[inv dT]

5' - GATCTACACGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNN--------- CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCGAT  - 3'
         XXXXX    XX XX  X XX                                                                             XX X  XX XX    XXXXX   
3' -  TAGCCAGACGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC ---------NNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCGCACATCTAG - 5'



Fill-in and Ligation:
               5' - GATCTACACGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCGAT
                        XXXXX    XX XX  X XX                                                                             XX X  XX XX    XXXXX   
                     TAGCCAGACGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCGCACATCTAG - 5'


Amplification and Sequencing
               5' - GATCTACACGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG NNNNNNNNNNNNNNNNNNNNNNNNNNNNN CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCGAT
									               		                3' - GACTCGCCCGACCGTTCCGTCTGGCNNNNNNNNNTAGAGCATACGGCAGAAGACGAAC - 5' - Amp  Primer 2
                                                                                             3' - GACAGAGAATATGTGTAGAGACTCGCCCGACCGTTCCG - 5' - Read 2 Primer
                                                                                             5' - CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG - 3' - Index Primer
AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAG - 3' Amp Primer 1
                        5' - GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG - 3' - Read 1 Primer


Bottom Strand (reverse of above)
	3' -  TAGCCAGACGGAACGGTCGGGCGAGTCTCTACACATATTCTCTGTC NNNNNNNNNNNNNNNNNNNNNNNNNNNNN GACAGAGAATATGTGTAGAGACTACCGCGCTCCCTCCGCACATCTAG - 5'


To order:
A1Top - 5' - GATCTACACGCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG - 3'
A1Bot - 5' - [Phos]CTGTCTCTTATACACA[ddC] - 3'
A2Top - 5' - [Phos]CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCGA[inv dT] - 3'
Amp Primer 1 - 5' - AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAG - 3' (This is the same as the normal nextera Kit; Double-check that this is correct with Shendure lab?)
Amp Primer 2 - 5' - CAAGCAGAAGACGGCATACGAGATNNNNNNNNNCGGTCTGCCTTGCCAGCCCGCTCAG - 3' (Where N is the barcode of choice)
Read 1 Primer - 5' - GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAG - 3'
Read 2 Primer - 5' - GCCTTGCCAGCCCGCTCAGAGATGTGTATAAGAGACAG - 3'
Index Primer  - 5' - CTGTCTCTTATACACATCTCTGAGCGGGCTGGCAAGGCAGACCG - 3'