GSM6754694: PMcut_scClone6_iPSC_IgG_CnR; Homo sapiens; OTHER
CUT&RUN was completed as previously described (Epicypher). In brief, iPS cells were harvested and then washed in wash buffer (20 mM Hepes KOH pH 7.5, 150 mM NaCl, 0.5 mM Spermadine, 1 Roche Complete Protease Inhibitor EDTA-free mini tablet per 10 mL) and bound to Concanavalin A beads (BioMagPlus) that had been activated and washed with binding buffer (20 mM Hepes KOH pH 8.0, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). The cells were then incubated with the Concanavalin A magnetic beads, primary antibody (either IgG (Sigma I8140) or H3K9me3 (Abcam ab8898), and antibody buffer (digi-wash buffer – 0.1% digitonin in wash buffer – with 2 mM EDTA) overnight at 4 C while rotating. Cells were washed with digi-wash buffer and then incubated in a solution containing protein A-MNase and digi-wash buffer for 10 minutes at room temperature. After incubation, the samples were washed in digi-wash buffer and 100 uL digi-wash buffer was added to the samples which were then placed on ice to chill for five minutes. After chilling, 2 uL of 100 mM CaC was added to activate protein A-MNase chromatin digestion. After 2 hours at 4 C with rotation, 100 uL of 2X stop buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% Digitonin, 50 ug/mL RNase A, 50 ug/mL Glycogen) was added to halt the reaction which was then incubated at 37 C for 30 minutes to release chromatin fragments. Supernatant was collected and DNA was extracted using phenol-chloroform and ethanol precipitation. The resulting DNA was quantified on a Qubit Fluorometer. CUT&RUN libraries were prepared for sequencing using the NEBNext Ultra II DNA Library Prep Kit (NEB #7103) according to manufacturer's protocol. Size selection of adaptor-ligated libraries was performed using AgenCourt Ampure XP beads (Beckman Coulter, A63881) according to the manufacturer's protocol. Size selection targeted <1 kb fragment size and libraries were amplified using 11 PCR cycles. Input amounts for library preparation using the NEBNext Ultra II DNA Library Prep Kit were 1 ng of purified libraries. Library quality and quantities were assessed using the Agilent DNA 1000 reagent kit (Agilent, 5067–1504) on the Agilent Bioanalyzer 2100 (Agilent, 5067–4626) and Qubit high sensitivity RNA assay kit (Thermo Fisher, Q32852), respectively before sequencing on NextSeq500 (Illumina)