title: GSM8791636: Mouse MuSC ASC PDS MicroC; Mus musculus; OTHER
library_construction_protocol: Micro-C for muscle stem cells was performed following published protocol for mammalian Micro-C. Briefly, cells were first crosslinked at 1 ml per million cells of 3mM DSG crosslinker (MedChemExpress, HY-114697) for 35 min at room temperature and then 1% formaldehyde was added and incubated for 10 more minutes. The crosslink was quenched with 0.375 M Tris(pH 7.0) for 5 min. Cells were pelleted at 1000g  at 4 °C for 5 min and resuspended with ice-cold PBS. 1 Million cells were split into each tube and pelleted by centrifugation. After snap frozen in liquid nitrogen, cells were kept at -80°C. For cell lysis, cells were thawed on ice for 5 min, and lysed with 0.5 ml MB 1 buffer (10 mM Tris–HCl, pH 7.5, 50 mM NaCl, 5 mM, MgCl2, 3 mM CaCl2, 0.2% NP-40, 1×PIC) on ice for 20 min, washed once with MB 1 buffer, then resuspended in 100 ul MB1 buffer. Mnase concentration for muscle stem cells was predetermined using MNase titration experiments. Chromatin was digested by adding 0.1 ul Mnase (NEB, M0247S) and incubating for 20 min at 37 °C, 1000rpm. Digestion was stopped by adding 8 μl of 500 mM EGTA and incubating at 65 °C for 10 min. Cells were then washed twice with ice-cold MB2 buffer (10 mM Tris–HCl, pH 7.5, 50 mM NaCl, 10 mM, MgCl2). The Mnase digested DNA ends were polished with T4 PNK (NEB M0201), followed by DNA polymerase Klenow fragment (NEB M0210), and then repaired and labeled by adding biotinylated dATP and dCTP (Jena Bioscience, NU-835-BIO14-S and NU-809-BIOX-S, respectively), and TTP/GTP. After washing with buffer MB3 (50 mM Tris–HCl, 10 mM, MgCl2 ). Ligation was performed for 4 h at room temperature using T4 DNA ligase (NEB M0202). Dangling ends were removed by a 15-minute incubation with Exonuclease III (NEB 0206) at 37 °C. DNAs were de-crosslinked overnight at 65°C. After DNA extraction by ethanol precipitation, size selection was performed using DNA purification beads to enrich ligated fragments at about 230bp. Then biotin selection was done using 10 μl Dynabeads MyOne Streptavidin C1 beads (Invitrogen 65001). For DNA library construction, a NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB, E7645S) was used according to the manufacturer's instructions.