GSM2533863: ChIP_NPC_H3K4me1_2; Mus musculus; ChIP-Seq
Cells were processed for FACS as described for HiC, except for permeabilization or fxCycle addition. GFP+ purified cells were immediately pelleted, frozen in liquid nitrogen and stored at -80C until further use. 1x10^6 cells per IP for chromatin marks, or 2.5x10^6 cells for CTCF/Ring1B were then thawed on ice, resuspended in cold cell lysis buffer (10mM Tris pH 8, 10mM NaCl, 0.2% NP-40) + 1xEDTA-free Protease Inhibitors. 10mM sodium butyrate was added if H3K27ac was examined. Cells were lysed for 20min, washed once with cold PBS and resuspended in 50ul per IP cold nuclei lysis buffer (50mM Tris pH8, 10mM EDTA, 1% SDS) + 1xProtease Inhibitors / Sodium Butyrate. Nuclei were lysed for 10min at 4C with rotation and then sonicated for 16-18 cycles using Bioruptor (Diagenode). After sonication, 5x volumes of IP dilution buffer (20mM Tris pH8, 2mM EDTA, 150mM NaCl, 1% Triton X-100, 0.01% SDS + protease inhibitors) was added, chromatin was precleared using 25ul Protein A dynabeads (ThermoFisher, Cat.N: 10002D) / 1mL for 1hrs at 4C with rotation.  Meanwhile, 25ul beads / IP were washed once with cold 0.5% BSA in PBS, and incubated with the antibody for 4-5hrs at 4C in 0.5ml 0.5% BSA in PBS). Beads were then washed once with 0.5% BSA in PBS, added to the precleared chromatin and incubated overnight at 4C with rotation. Beads were then washed once with cold IP wash buffer 1 (20mM Tris pH8, 2mM EDTA, 50mM NaCl, 1% Triton X-100, 0.1% SDS), twice with high salt wash buffer (20mM Tris pH8, 2mM EDTA, 500mM NaCl, 1% Triton X-100,0.1% SDS), once with cold IP wash buffer 2 (10mM Tris pH8,1mM EDTA,250mM LiCl,1% NP-40,1% sodium deoxycholate) and twice with cold TE buffer (1mM Tris pH8, 1mM EDTA). DNA:protein complexes were then eluted twice for 15min at 65C in 100ul elution buffer (100mM NaHCO3, 1%SDS) each time. 16ul 5M NaCl was then added and samples + inputs were reverse cross-linked at 65C, RNase A and proteinase K treated and purified using ultrapure phenol/chloroform (ThermoFisher, Cat.N: 15593-049). Libraries were prepared using Illumina's TruSeq ChIP Sample Preparation Kit, according to the manufacturer's instructions with minor modifications. In brief, 5-10ng of ChIP DNA was end repaired, 3' Ends were adenylated and Illumina adapters were ligated. Libraries were then PCR amplified using KAPA HiFi Library Ampification Kit for 10-12 cycles to maximize complexity and the gel-based size-selection was performed after PCR amplification.