GSM1462468: H3K27ac_REP2_ChIPSeq; Homo sapiens; ChIP-Seq
Tfh cell sorts were initially gated on CD4+CD19-, then CD45RO+, then as CXCR5hi (GC Tfh). The following anti-human Abs were used: CD45RO (clone UCHL1), CD19 (clone HIB19), CD4 (clone RPA-T4) (eBioscience, San Diego, CA); CXCR5 (clone RF8B2). GC Tfh (CXCR5hi PD1hi CD45RO+ CD4+ T cells, CD19‑) were isolated from human tonsils, fixed with 1% formaldehyde, lysed and sonicated to generate fragments less than 500bp. Chromatin immunoprecipitation was performed by incubation of the chromatin with antibodies against BCL6 (Santa Cruz, sc-858), H3K4me3(Abcam, ab8580), H4K4me1(Abcam, ab8895) and H3K27ac (Abcam, ab4729). Immunocomplexes were pulled down using protein A beads and after increasing stringency washes 10ng of ChIP DNA was recovered and used to generate a BCL6 ChIP-seq library according to Illumina ChIP-seq Library preparation Kit. A negative control library was prepared in parallel using 10ng of input chromatin DNA. Libraries were quantified and validated using Agilent Technologies 2100 Bioanalyser for size, concentration and purity. Both libraries were sequenced using HiSeq 2000 for 50 cycles.