GSM8689877: Liver Prox1 rep 3; Mus musculus; OTHER
Cells were harvested with Accutase and strained through a 70 um strainer followed by CUT&RUN processing. 250,000 cells were washed twice with wash buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, and 1X Roche Complete Protease Inhibitor), then resuspended in wash buffer and added to Concanavalin-A beads (Polysciences) pre-activated in cold binding buffer (20 mM HEPES-KOH pH 7.5, 10 mM KCl, 1 mM CaCl2, and 1 mM MnCl2). The bead-cell suspension was rotated (RT, 10 min). The supernatant was removed on a magnet and the beads were resuspended in antibody buffer (0.2 mM EDTA, 0.05% w/v digitonin in wash buffer). Primary antibody (as indicated) or control (rabbit IgG) was added at 1:100 v/v, and cells were incubated on a nutator (overnight, 4°C). Beads were washed twice in digitonin-wash buffer (0.05% w/v digitonin in wash buffer), resuspended in 700 ng/mL pAG-MNase (Protein Expression and Purification Core Facility, EMBL, Heidelberg) in digitonin-wash buffer, and rotated (4°C, 1 h). pAG-MNase-loaded beads were then washed twice in digitonin-wash buffer, resuspended in digitonin-wash buffer, and placed on ice. 1 uL of 100 mM CaCl2 was added to induce chromatin digestion, and the mixture was incubated on ice (30 min). 50 uL of 2x stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% w/v digitonin, 50 ug/mL RNase A, 50 ug/mL glycogen, 0.5 ng/mL spike-in E. coli DNA) was added, and the suspension incubated at 37°C for 10 minutes to release chromatin fragments from cells. The supernatant was subjected to phenol-chloroform extraction, and purified DNA fragments were used for library preparation with NEBNext DNA Library Prep Kit for Illumina (NEB E7645). Libraries were then sequenced (paired-end, 2x40 bp) on the NextSeq 550 platform (Illumina).