title: GSM8763488: GFP-KLF2+ LCM, IgG, rep 1; Mus musculus; OTHER
library_construction_protocol: CUT&RUN was performed as previously described in (Skene, Henikoff and Henikoff, 2018) with modifications.  Cells were permeabilized with wash buffer containing 0.05% w/v Digitonin (Sigma-Aldrich). Each sample was incubated at 4°C with anti-GFP (Abcam, clone 290) or rabbit IgG (Sigma) at a concentration of 50μg/mL and left to rock overnight. Permeabilized cells were washed and incubated rotating at room temperature for 10 min with pA-MNase (kindly provided by the Henikoff lab) at a concentration of 700ng/mL. After washing, cells were incubated at 0°C and MNase digestion was initiated by addition of CaCl2 to 1.3mM. After 2h, the reaction was stopped by the addition of EDTA and EGTA. Chromatin fragments were released by incubation at 37°C for 10 min. DNA was purified using DNA Clean&Concentrator-5 kit (Zymo). Libraries were prepared using the SMART-seq2 protocol (Picelli et al., 2014), quantified by Qubit (ThermoFisher), and quality checked by Bioanalyzer (Agilent) before sequencing on an Illumina NovaSeq as paired-ends to a depth of 3-5 million.