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'