GSM7727555: CAF,231EV,H3K4me3,ChIP; Mus musculus; ChIP-Seq
CAF cells (5 × 106) plated into a 10 cm dish were cross-linked for 10 min at room temperature with 1% fina formaldehyde and then quenched with 0.125M of glycine for 5 min. Aspirated off the supernatant and adde PBS to wash the cells. The cross-linked cells were harvested with PBS and then centrifuged for 5 min at 500 ×g. Supernatant was discarded and the precipitates were lysed with lysis buffer (50 mM Tris-HCl, pH 8.0, 0.5 SDS, 5 mM EDTA). After centrifugation at 10,000 × g for 2 min at 4°C, the pellet was washed once with digestion buffer (50 mM Tris-HCl, pH 7.6, 1 mM CaCl2, 0.2% triton X-100), incubated in 500 μL digestion buffer with 3 μL MNase (NEB, M0247S) at 37°C for 20 min and quenched with 5 μL of 0.5 M EDTA. Samples were sonicate briefly and the supernatant was transferred to new tubes after centrifugation. ChIP-seq libraries were constructed with ChIP and input DNA using VATHS Universal DNA Library Pre Kit for Illumina (Vazyme, ND606). Briefly, 50 μL of DNA (8-10 ng) was end-repaired for dA tailing, followed by adaptor ligation. Each adaptor was marked with a barcode of 8 bp DNA. Adaptor-ligated DNA was purified by AMPure XP beads (1:0.6) and then amplified by PCR of 9 cycles with the primer matching with adaptor universal part. Amplified DNA was purified again using AMPure XP beads (1:0.9) in 20 μL EB elution buffer. Fo multiplexing, libraries with different barcode were mixed with equal molar quantities (30-50 million reads per 560 library).