title: GSM6203891: Ki67_aid_HCT116_IAA_mitosis_ChIP_input_r1; Homo sapiens; ChIP-Seq
library_construction_protocol: 1. pA-DamID: 1 million permeabilized cells were processed with pA-DamID (van Schaik, 2020) with a primary antibody and for a Dam control condition, to specifically deposit m6A marks at chromatin nearby the primary antibody. Genomic DNA was isolated using the Bioline Isolate II Genomic DNA Kit; 2. RNA-seq: RNA was isolated using the Qiagen RNeasy column purification kit; 3. Repli-Seq: cells were harvested as for pA-DamID and immediately fixed in ice-cold ethanol. 4. ChIP-seq: Cells were crosslinked (1% FA, 10 minutes) and quenced with glycine (2.0M). 5. Hi-C: Cells were harvested and crosslinked (2% FA, 10 minutes). 1. pA-DamID: the full protocol is described in Leemans, 2019, except that the DpnII digestion is omitted. 500 ng of genomic DNA was digested at m6A-methylated GATC sequences with DpnI and ligated to adapters. DNA fragments were amplified and further processed with end-repair, 3A-overhang tailing and Y-shaped adapter ligation, before a final round of 10-12 PCR cycles with illumina index adapters; 2: RNA-seq: sequencing libraries were prepared using the Illumina TruSeq polyA stranded RNA kit; 3. Repli-seq: replicated DNA was enriched for with a BrdU IP, before libraries were prepared as described in Marchal, 2018; 4. ChIP-seq: Chromatin was fragmented to ~300 bp fragments with a Bioruptor Plus sonication device (Diagenode). Ki-67 antibody (ab15580, Abcam; 5 μL per ChIP) was coupled with Protein G beads (Thermo Fisher Scientific) and incubated with fragmented chromatin overnight at 4 °C. Released DNA fragments were purified with the MinElute PCR Purification kit (Qiagen). The purified DNA fragments were prepared with the KAPA HTP Library Preparation kit (Roche); 5. Hi-C: Cross-linked nuclei were digested with MboI and biotinylated nucleotides incorporated in the overhangs. Following blunt-end ligation, the ligated fragments were extracted using streptavidin pulldown and prepared for sequencing using a standard end-repair / 3A-tailing procedure.