Bisulfite-Seq analysis of RT sample TARGET-52-PASGGN-01A-01D
Bisulfite-Seq analysis of rhabdoid tumor sample TARGET-52-PASGGN-01A-01D using Illumina HiSeq 2500
To track the efficiency of bisulfite conversion, 1ng lambda DNA (Promega) was spiked into 1µg genomic DNA quantified using Qubit fluorometry and arrayed in a 96-well microtitre plate. DNA was sheared to a target size of 300bp using Covaris sonication and the fragments end repaired using DNA ligase and dNTPs at 30oC for 30min. Repaired DNA was purified using a 2:1 AMPure XP beads to sample ratio and eluted in 40µL elution buffer in preparation for A-tailing; the addition of adenosine to the 3’ end of DNA fragments using Klenow fragment and dATP incubated at 37oC for 30min. Following reaction clean-up with magnetic beads, cytosine methylated paired-end adapters (5’-AmCAmCTmCTTTmCmCmCTAmCAmCGAmCGmCTmCTTmCmCGATmCT-3’ and 3’-GAGmCmCGTAAGGAmCGAmCTTGGmCGAGAAGGmCTAG-5’) were ligated to the DNA at 30oC, 20min and adapter flanked DNA fragments bead purified. Prior to bisulfite conversion an aliquot of library fragments was amplified with 10 cycles of PCR and sized on an Agilent Bioanalyzer High Sensitivity DNA chip. Amplicons were observed between 200-700bp in length. Bisulfite conversion of the methylated adapter-ligated DNA fragments was achieved using the EZ Methylation-Gold kit (Zymo Research) following the manufacturer’s protocol. Five cycles of PCR using HiFi polymerase (Kapa Biosystems) was used to enrich the bisulfite converted DNA and introduce fault tolerant hexamer barcode sequences. Post-PCR purification and size-selection of bisulfite converted DNA was performed from pre-cast 8% TBE gels (Invitrogen), extracting the 350-500bp fraction, or 275-425bp fraction if the former was of weak intensity. Gel slurries were added to Spin-X filter tubes (Fisher) and the eluate ethanol precipitated and resuspended in EB. To determine final library concentrations fragment sizes were assessed using a high sensitivity DNA assay (Agilent) and DNA quantified by Qubit fluorometry. Where necessary, libraries were diluted in elution buffer supplemented with 0.1% Tween-20 to achieve a concentration of 8nM for Illumina HiSeq2000/2500 flowcell cluster generation.
BCCAGSC
A48363-1