title: GSM5384963: H3K4me3 ChIP-seq 1 run1; Mus musculus; ChIP-Seq
library_construction_protocol: mESCs were cross-linked for 5 minutes with 1% formaldehyde and quenched with 0.25μM glycine. Chromatin fragmentation was performed in sonication buffer (20mM Tris pH 8.0, 2mM EDTA, 0.5mM EGTA, 0.5% SDS, 0.5mM PMSF) with a Qsonica Q800R bath sonicator at 70% amplitude pulsing with 15 second “ON” and 45 seconds “OFF” cycles for 30 minutes total sonication time. Fragment sizes were checked by agarose gel. Fragmented chromatin from 7.5M mESCs was used as input for immunoprecipitation. ChIP material was diluted with 1mL of IP buffer (20mM Tris pH 8.0, 2mM EDTA, 0.5% Triton X-100, 150mM NaCl, 10% Glycerol, 5% BSA) and pre-cleared with 10μL DynaBeads Protein A (ThermoFisher) for 1 hr at 4°C. The supernatant was then further diluted with 250μL IP buffer and incubated with 16 μL H3K4me3 antibody (EpiCypher Cat#13-0028) overnight at 4°C. Bound chromatin was isolated with 150 μL of pre-blocked DynaBeads Protein A for 2 hrs at 4°C. Beads were washed 1x in low salt (20mM Tris pH 8.0, 2mM EDTA, 1% Triton X-100, 150mM NaCl, 0.1% SDS), 3x in high salt (20mM Tris pH 8.0, 2mM EDTA, 1% Triton X-100, 500mM NaCl, 0.1% SDS), 1x in LiCl (20mM Tris pH 8.0, 2mM EDTA, 250mM LiCl, 1% IGEPAL, 1%[w/v] sodium deoxycholate), and 2x in TE (10mM Tris pH 8.0, 0.1mM EDTA) prior to elution with 2x 250uL of elution buffer (10% SDS, 100mM sodium bicarbonate). Crosslinks were reversed by the addition of 0.2M NaCl and incubation at 65°C overnight. The IP mixture was treated with Proteinase K (Invitrogen) for 1 hr at 50°C. DNA was extracted with phenol:chloroform:isoamyl alcohol and precipitated with ethanol. Sequencing libraries were prepared using the NEBNext Ultra II DNA Kit (New England Biolabs) as per the manufacturer's protocol.