GSM1448241: 5284_CD8 [Bisulfite-Seq]; Homo sapiens; Bisulfite-Seq
Genomic DNA was prepared from cell pellets using an All Prep kit (QIAGEN).  DNA was bisulfite converted using the EpiTect Bisulfite conversion kit (QIAGEN). Bisulfite treated genomic DNA was amplified by microdroplet PCR amplification on an RDT 1000 (RainDance Technologies) with a custom primer library according to the manufacturer’s instructions.  Briefly, microdroplets containing genomic DNA were merged with microdroplets from the custom primer droplet library.  The custom primer droplet library consists of a collection of individual primer droplets, where each primer droplet contains matched pairs of forward and reverse primer (for each amplicon defined within the primer library.  For primers covering a CpG, a degenerate base (C/T or G/A, depending on the strand) was used at that location. The RDT1000 generated each PCR droplet by pairing a single genomic DNA template droplet with a single primer droplet.  Paired droplets flow past an electrode embedded in the chip and are instantly merged together. All resulting PCR droplets were automatically dispensed as an emulsion into a single PCR tube and transferred to a standard thermal cycler for PCR amplification.  Each sample generated more than 1 million singleplex PCR droplets. Samples were amplified for 55 cycles, the droplet emulsion was broken, and the PCR product was purified over a MinElute column (QIAGEN) following the manufacturer’s recommended protocol. RainDance PCR products were end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. End-repaired products were concatenated using T4 DNA ligase and then fragmented to 200 bp using a Covaris S2. Fragmented PCR product was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment and dATP to yield a protruding 3- 'A' base for ligation of adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified for 15 cycles and library fragments of ~350 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on an Illumina HiSeq 20002000  following the manufacturer's protocols.  100 bp single-end reads were generated with 10 samples per lane.