GSM5133470: DT-40 and mitotic Hela S3 treated with CAY10683 cells (1:3 ratio) - H3K9ac_rep1; Gallus gallus; Homo sapiens; ChIP-Seq
Hela S3 cells (unsynchronized or STC synchronized with or without the indicated treatments) and unsynchronized and untreated DT-40 cells were cross-linked and mixed. The cell mixture was lysed and sonicated. Immunoprecipitation, using anti-H3K9ac (C5B11, Cell Signalling Technology), was carried out with inverting at 4°C for 14–16 h. Antibody-chromatin complexes were pulled-down using Protein A/G magnetic beads (Dy-10001D and Dy-10003D, Invitrogen), washed and then eluted. After cross-link reversal and Proteinase K treatment, immunoprecipitated DNA was extracted with 1.8x Agencourt AMPure XP beads (BCA63881, Beckman Coulter). ChIP DNA (or unenriched whole cell extract) were prepared for Illumina sequencing as described (Yehuda, Blumenfeld et al., 2018). Briefly, DNA was subjected to a 50 μl end repair reaction, cleaned by 1.8× Ampure XP beads, followed by a 50μl A-tail reaction. The products were cleaned and ligated to 0.75 μM illumina compatible forked indexed adapters. Ligation products were size selected in order to remove free adaptors. Ligation products were amplified with 15 (Input DNA) or 18 PCR cycles (ChIP DNA). Amplified DNA was size selected for 300–700 bp fragments using Ampure XP beads. The final quality of the library was assessed by Qubit and TapeStation. Libraries were pooled and sequenced on NextSeq (Illumina), using a paired-end protocol