CD8+ T cels were enriched from tissue using negative selection magnetic beads. DNA were extracted using QIAGEN Dneasy, as per manufacturer's recommendations. Following enzymatic digestion and sonication, immunoprecipitation was performed using anti-5meC (Eurogentek, BI-MECY-0100, lot 081103). Methyl-seq library construction was performed with NEXTflex Methyl-seq kit (Biooscientific, Austin, TA) per manufacturer’s recommendations. Briefly, 1200 ng of genomic DNA was sheared to an average size of 200-400bp with the Covaris S2 (Covaris, Woburn, MA). Sheared DNA sizing was confirmed with a Bioanlayzer 2100 (Agilent, Santa Clara, CA) and subsequent end repair, adenylation, and adaptor ligation was performed. DNA was heated to 95°C, and incubated with anti-5methylcytosine antibody (Epigentek, Farmingdale, NY). Methylated DNA was immunoprecipitated with Dynabeads (Life Technologies, Grand Island, NY), and released from the beads using Proteinase K digestion. DNA was purified using the MinElute PCR Purification Kit (QIAGEN), followed by library PCR amplification and gel size selection.
GEO Accession
GSM1465121