title: GSM4850099: HMLE KO MaSC H3K27me3 rep2; Homo sapiens; OTHER
library_construction_protocol: Cut&Run was performed as described previously (Skene et al. 2018). Briefly, 4-5*105 HMLE cells, together with 0.25*105 D. melanogaster Schneider 2 cells, were washed twice with Wash buffer (20 mM HEPES pH=7.5, 150 mM NaCl, 0.5 mM spermidine) supplemented with protease inhibitors. Cells were then resuspended in Binding buffer (20 mM HEPES pH=7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) containing 10 µL of blocked BioMag®Plus Concanavalin A-coated beads (Polysciences, ref. 86057). After 10 min at RT, cells:beads were transferred to 50 µL of Antibody solution (1:100 dilution of primary antibody in Wash buffer plus 0.1% digitonin [Millipore, ref. 300410-5GM]) and incubated for 1 h at RT on an end-to-end rotator. Cells:beads were washed three times with Wash buffer-0.1% digitonin and incubated for 10 min with pA-MNase diluted in Wash buffer-0.1% digitonin (produced by the Protein Expression and Purification Core Facility of Institut Curie). After three additional washes in Wash buffer-0.1% digitonin, tubes were placed in an ice/water bath and equilibrated to 0°C for 10 min. To trigger digestion of the DNA, CaCl2 was added (2 mM final concentration) for 30 min. Digestion was stopped by addition of 2X STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% digitonin, 1:200 RNase A [50 μg/mL final concentration]). Digested DNA fragments were released from cells by incubating sample tubes on a heat block at 37 °C for 10 min. The supernatant was then recovered by placing tubes on a magnetic stand and DNA was purified using the NucleoSpin Gel and PCR Clean‑up kit (Macherey Nagel, ref. 740609). Cut&Run-Seq libraries were prepared using the Accel-NGS 2S Plus DNA library kit (Swift Biosciences, ref. 21096) according to manufacturer's instructions, and sequenced on an Illumina Novaseq 6000 (PE100) at the Institut Curie Next Generation Sequencing Core Facility.
attributes.tag: GEO Accession
attributes.value: GSM4850099