GSM5708873: M15_KTSminus_rep1; Mus musculus; OTHER
CUT&RUN was performed Mouse M15 and M15 KTS minus cells. Cells were gently washed twice in room temperature Wash buffer [20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine and a Roche complete EDTA-free tablet (Sigma-Aldrich) per 50 ml], with 3 min centrifugation at 600 xg, mixed with activated Concanavalin A-coated magnetic beads (Bangs Laboratories) and rotated 5-10 min. Beads were captured by placing on a magnet stand, decanted and resuspended in Antibody buffer [2 mM EDTA and antibody at 1:100 in Dig-wash (Wash buffer supplemented with 0.05% digitonin (Calbiochem)]. Antibodies used were Anti-Wilms Tumor Protein antibody (Abcam, ab89901) and IgG (IgG (Bethyl, A60-111A guinea pig anti-rabbit). After overnight binding at 4 oC with rotation, beads were captured and washed once or twice in Dig-wash and incubated 1 hr at 4 oC and washed once in Dig-wash. Beads were resuspended in Protein A-MNase (kindly provided by Steven Henikoff lab, 360 µg/ml) 1:500 in Dig-wash, incubated 1 hr at 4 oC, washed twice in Dig-wash and resuspended in 100 µL Dig-wash. Tubes were placed 0 oC, mixed with 2 µL 100 mM CaCl2, incubated at 0 oC for 30 min, and reactions were stopped by addition of 100 µL 340 mM NaCl, 20 mM EDTA pH8, 4 mM EGTA, 0.05% digitonin, 50 µg/ml glycogen and 200 pg mono-nucleosomal S. cerevisiae (spike-in) DNA. Samples were incubated 10 min at 37 oC and centrifuged 5 min 16,000 xg at 4 oC and the supernatant was treated with 25 µg/ml RNAse A (Thermo) 10 min at 37 oC. After phenol-chloroform-isoamyl alcohol and chloroform extraction, DNA was precipitated by addition of 2.5 volumes 100% ethanol, chilled on ice, centrifuged 10 min at 4 oC at 16,000 xg and the pellets were rinsed in 100% ethanol and air-dried. Pellets were resuspended in 1 mM Tris pH8 0.1 mM EDTA and used for standard Illumina library preparation. Libraries were prepared with NEBNext Ultra II DNA Library Prep Kit for Illumina (E7645S), according to the manufacturer's instructions. After adapter ligation, DNA was PCR amplified with Illumina primers for 14 cycles. The libraries were sequenced on Illumina sequencing PE150-Xten.