GSM7093562: CUT&RUN - KO15_d2 - IgG; Mus musculus; OTHER
CUT&RUN was performed using the CUTANATM KIT (Epicypher, 14-1048) according to the manufacturer's instructions with minor modifications (33,34). Briefly, 5 x 105 cells were collected as a single cell suspension and washed three times with 100 μl/sample wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine and 1 Roche complete tablet/50ml). Cells were then bound to activated Concanavalin A beads and incubated for 10 minutes at RT, followed by incubation with 0.5 μg anti-H3K27me3 (Invitrogen, MA5-11198) antibody or IgG (Epicypher, 18-1401) diluted in antibody buffer on a nutator overnight at 4°C. After 3 washes of magnet-bound beads with digitonin buffer, pAG/MNase was added and incubated for 10 minutes at RT, washed and resuspended in digestion buffer for a 30-minute incubation on ice. The reaction was stopped by addition of STOP buffer and incubated for 10 minutes at 37 °C. The supernatant containing enriched DNA was then extracted using the supplied DNA Clean-up Columns. Libraries were prepared using the NEBNext® Ultra II DNA library Prep Kit for Illumina (New England Biolabs®, NEB E7645L) according to Epicypher's instructions, and amplified by PCR using NEBNext Dual Index Primers (New England Biolabs, E6440S) with the following settings: 98°C 45 sec; 14 x 98°C 15 sec, 60°C 10 sec; 72°C 1 min. Library concentration was quantified using Qubit HS dsDNA Quant kit (ThermoFisher, Q32851).Quality and size distribution were further assessed on an Agilent 2100 Bioanalyzer using a High Sensitivity DNA Analysis Kit (Agilent, 5067-4626). Libraries were diluted to 4nM and pooled together for PE50 sequencing on NextSeq500 or NextSeq2000 for minimally 5 million reads per sample.