GSM9688187: PBS_CD4_Rep3; Mus musculus; Bisulfite-Seq
Splenocytes were stained with antibodies against CD4 and CD8 and sorted using BD Symphony S6. Sorted CD4+ and CD8+ T cells were collected into flow buffer for further downstream epigenetic analysis. DNA isolation was performed on sorted T cells using Puregene Cell kit (Qiagen) according to manufacturer's guidelines. Zymo Clean and Concentrator Kit was used to clean up the collected DNA. DNA concentration was measured using Qubit Fluorometer 1X high sensitivity assay, and the DNA quality was measured using Nanodrop. 100ng DNA was used per sample. A 2 hour digestion using Mspl restriction enzyme (new England biolabs) was perfomed to enrich for CpG dense regions. Library preparation was performed using NEBNext Ultra II Modules (New England Biolabs) using methylated adaptors. Ampure XP magnetic beads (Beckman Coulter) were used to obtain an average size of approximately 200bp from the DNA fragments. Next, we used the EZ DNA Methylation-Gold Kit (Zymo Research) to perform bisulphite conversion. PCR amplification of DNA was performed using Q5U polymerase and NEBNext Multiplex Oligos for Illumina (New England Biolabs). After quantifying the libraries using Qubit fluorometer, they were normalized and sequenced at the OHSU MPSSR sequencing core.