title: GSM4586751: AG35_CD4; Chlorocebus pygerythrus; Bisulfite-Seq
library_construction_protocol: To assess CpG methylation across the 20kb region encompassing CD4 regulatory regions, genomic DNA from CD4+, CD4-CD8aa+, and CD4-CD8ab+ T cells of cryopreserved AGM splenic cell suspensions was isolated using the Qiagen allprep DNA/RNA isolation kit (Qiagen). At least 3ug of genomic DNA was sheared with a Covaris sonicator (Woburn, MA) to 100-175nt fragment sizes. Library preparation was then followed in exact accordance to the Agilent methylseq library prep protocol.  In brief, DNA libraries were prepared first by repairing and dA-tailing the 3' end of the DNA fragments.  Methylated adapters were subsequently ligated onto the DNA fragments.  Genomic DNA libraries were then hybridized with proprietary hybridization probes designed by Agilent (Santa Clara, CA) to be complimentary to the 20kb capture region of the CD4 locus and incubated overnight at 65 C.  Hybridized fragments were then captured with streptavidin beads, washed, and subsequently deaminated with the EZ DNA methylation lightning kit (Zymo Research).  Bisulfite-converted libraries were indexed, amplified with 12 PCR cycles and subsequently pooled for sequencing on an HTseq 4000 (Illumina).