GSM7039213: K562, H3K4me3; Homo sapiens; ChIP-Seq
MChIP-C: Cells were crosslinked for 10 min at room temperature in fresh full growth medium supplemented with 2% formaldehyde. Crosslinked cells were permeabilized in PBS supplemented with 0.01% digitonin on ice, chromatin was subjected to 1h digestion with 35-40 U of micrococcal nuclease (Thermo Scientific, EN0181). Next, DNA ends were repaired with 100 U of T4 Polynucleotide Kinase (NEB, M0201L) and 50U of Klenow fragment (NEB, M0210L). Blunted DNA ends were proximity ligated with 75U of T4 DNA ligase (ThermoScientific, EL0012). Chromatin was sheared on ice with a VirSonic 100 cell disrupter with 3 15-sec pulses on “15” power setting, separated with 3 min periods of recovery, transferred to RIPA buffer and immunoprecipitated with anti-H3K4me3 antibodies (Active motif, 39016) and protein A/G magnetic beads (Thermo Scientific, 26162). DNA was reverse-crosslinked and isolated with phenol-chloroform extraction and ethanol precipitation. ChIP-seq:  Cells were crosslinked for 10 min at room temperature in fresh full growth medium supplemented with 2% formaldehyde. Crosslinked cells were permeabilized in PBS supplemented with 0.01% digitonin on ice. Permeabilized cells were resuspended in ice cold ChIP lysis buffer (50 mM Tris-HCl pH 8.0, 1% SDS, 10 mM EDTA), chromatin was sheared with 4 30-sec ultrasound pulses on “15” power setting of VirSonic 100 sonicator. Incubation with antibodies, immunoprecipitation, DNA isolation were performed as described in MChIP-C protocol. MChIP-C and ChIP-seq NGS libraries were prepared from immunoprecipitated DNA with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645) according to the manufacturer's recommendations, with the following modifications: 1) we decreased the reaction volumes two-fold; 2) instead of the adaptor included in the kit, we used 1 μL of barcoded Y-shaped Illumina TruSeq adapters (Illumina, 20015960 and 20015961) per reaction and extended ligation time to 1h.  We used either 17 μL (~0.37×) (for MChIP-C) or 35 μL (0.76×) (for ChIP-seq) of AMPure XP beads (Beckman Coulter, A63881) in the pre-PCR clean-up. Adapter-ligated material was amplified with 12-14 cycles of PCR using KAPA HiFi HotStart PCR Kit (Roche, 07958897001) and universal P5/P7 Illumina primers. OTHER:MChIP-C, ChIP-seq