H3K4me3 CUT&RUN profiling in wild-type mouse embryonic stem cells (mESCs)
H3K4me3 CUT&RUN profiling in wild-type mouse embryonic stem cells (mESCs)
ESCs were grown in incubators at 37 °C with 5% CO2 in gelatin-coated tissue culture dishes. The culture medium was composed of Glasgow minimum essential medium (GMEM; Gibco cat. 11710035) supplemented with 15% foetal bovine serum (batch tested), 1x L-glutamine (Gibco cat. 25030024), 1x MEM non-essential amino acids (Gibco cat. 11140035), 1 mM sodium pyruvate (Gibco cat. 11360039), 0.1 mM 2-mercaptoethanol (Gibco cat. 31350010)  ESCs were grown in Serum/LIF media before harvesting. Serum/LIF: Glasgow minimum essential medium (GMEM; GIBCO ref. 11710035) supplemented with 15% fetal bovine serum (FBS; GIBCO ref. 10270106, batch tested), 100U/ml leukemia inhibitory factor (LIF; Sigma Aldrich ref. ESG1107, batch tested), 1x L-glutamine (GIBCO ref. 25030024), 1x MEM non-essential amino acids (GIBCO ref. 11140035), 1mM sodium pyruvate (GIBCO ref. 11360070) and 0.1mM 2-mercaptoethanol (GIBCO ref. 31350010). Cells were grown on gelatine-coated dishes at 37°C and 5% CO2. The EpiCypher CUTANA ChIC/CUT&RUN kit (v3.1, Stratech ref. 14-1048-EPC) was used for target DNA extraction and followed without modification. As input material, 500,000 cells per genotype were counted out and used. Libraries for next-generation sequencing were generated using the KAPA HyperPrep Kit (v7.19, Roche ref. 07962347001) with KAPA dual-indexed adapters (Roche ref. 08278555702) following the manufacturer's protocol with the following modifications to account for the low DNA yield from CUT&RUN: For each reaction, 5 ng of DNA was used as input. End-repair and A-tailing were carried out at 20°C for 30 min and 58°C for 45 min (modified from 65°C) to prevent melting of small fragments. Adapters were diluted to 1.5 µM and ligated with DNA fragments, with incubation extended to 30 min (modified from 15 min) at 20°C to improve conversion efficiency. DNA was then cleaned with the described 0.8X KAPA beads, but after that eluted in 55 µl EB (Qiagen ref. 19086) and subjected to an additional 1.1X bead cleanup. For both, DNA was bound to beads for 15 min and beads dried for 5 min before elution. Library amplification was performed with a modified cycling program to enrich small fragments as follows: Initial denaturation at 98°C for 45 sec; 14 cycles of 98°C for 15 sec and 60°C for 10 sec; final extension at 72°C for 1 min; and storage at 4°C. Amplified libraries underwent two further cleanups (1X bead cleanup per protocol, followed by an additional 1.1X step) before final elution in 22 µl EB. Libraries were quantified using the Qubit system (Invitrogen ref. Q32851), and insert sizes were assessed with an Agilent 2100 Bioanalyzer and High-Sensitivity DNA kit (Agilent ref. 5067-4626).