GSM6235125: GM21, Day 32, Rep 2, K4me1, ActiveMotif; Homo sapiens; ChIP-Seq
Immunoprecipitation was performed overnight at 4 °C with rotation using 1 × 106 cell equivalents per immunoprecipitation using antibodies (5 µg) against H3K4me1 (Active Motif, Carlsbad, MA; Cat no. 39297) and H3K27ac (Active Motif, Carlsbad, MA; Cat no. 39133). Subsequently, 30 µl of Ultralink Resin (Thermo Fisher Scientific, Waltham, MA) was added and allowed to tumble for 4 h at 4 °C. The resin was pelleted by centrifugation and washed three times in low-salt buffer (150 mM NaCl, 0.1% SDS, 1% Triton X-100, 20 mM EDTA, 20 mM Tris-HCl pH 8.0), one time in high-salt buffer (500 mM NaCl, 0.1% SDS, 1% Triton X-100, 20 mM EDTA, 20 mM Tris-HCl pH 8.0), two times in lithium chloride buffer (250 mM LiCl, 1% IGEPAL CA-630, 15 sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0) and two times in TE buffer (10 mM Tris-HCl, 1 mM EDTA). Washed beads were resuspended in elution buffer (10 mM Tris-Cl pH 8.0, 5 mM EDTA, 300 mM NaCl, 0.5% SDS) treated with RNAse H (30 min, 37 °C) and Proteinase K (2 h, 37 °C), 1 µl glycogen (20 mg/ml; Ambion, Austin, TX) was added and decrosslinked overnight at 65 °C. For histone modifications, DNA was recovered by mixing the decrosslinked supernatant with 2.2× SPRI beads followed by 4 min of incubation at room temperature. The SPRI beads were washed twice in 80% ethanol, allowed to dry, and DNA was eluted in 35 µl of 10 mM Tris-Cl pH 8.0. Libraries were constructed using an Accel-NGS 2S Plus DNA Library kit (21024; Swift Biosciences, Ann Arbor, MI) and amplified for 9 cycles. Libraries were then resuspended in 20 µl of low EDTA–TE buffer. Libraries were quality controlled in an Agilent Technologies 2100 Bioanalyzer (Applied Biosystems, Santa Clara, CA) and quantified using an Invitrogen Qubit DS DNA HS Assay kit (Q32854) (Thermo Fischer Scientific, Waltham, MA).