GSM6268271: H3K9me3.ZNF695KD.BJ.R3; Homo sapiens; OTHER
CUT&RUN was performed as described in (Meers et al. 2019a). An interactive version of this protocol is available on protocols.io at dx.doi.org/10.17504/protocols.io.zcpf2vn. The following modifications were made: 500,000 BJ fibroblast cells per replicate were used. Accutase was used to dissociate cells. The Wash Buffer was altered to final concentrations of 20mM HEPES pH 7.5, 0.5mM spermidine, 75 mM NaCl, and Roche Complete Protease Inhibitor EDTA-free. The digitonin concentration was altered to 0.05% for the Dig-wash buffer, the Antibody buffer and the 2x Stop buffer. Binding of primary antibodies was always carried out overnight. For H3K9me3, the antibody used was Abcam ab8898. For V5, the antibody used was ThermoFisher Ref#46-0705, Lot# 2378586. Secondary antibodies and associated wash steps were not used for histones. When binding the protein A/G-MNase fusion protein only 50uL of DIG-wash buffer and MNase was used. The standard chromatin and digestion option 1: standard CUT&RUN protocol was used.  During the elution of CUT&RUN fragments from insoluble nuclear fragments the elution was carried out for 20 minutes. The final samples were resuspended in 30uL of 0.1x TE. The libraries were prepped using the following protocol: Nan Liu 2021, Library Prep for CUT&RUN with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (E7645) found at protocols.io: https://dx.doi.org/10.17504/protocols.io.bagaibse. The protocol was also described in (Liu et al. 2018) and is optimized for small fragments 25-150 bp. 15 µL of each sample was used for library preparation. 13 cycles of PCR amplification were used. The first round of size selection during cleanup of PCR amplification was skipped for H3K9me3 samples and 30uL of AmpureXP beads were used for the second round of size selection for histone profiles. Library profiles were checked using the Agilent High Sensitivity DNA TAPESTATION and library size was determined. Library concentrations were determined using Qubit. Diluted libraries were denatured in 0.2 M NaOH, loaded into the cartridge of the NextSeq 500/550 High Output v2 kit (Illumina FC-404-2005, 75 cycles) at a concentration of 3.2 pM in the kit's Hybridization Buffer, and sequenced in an Illumina NextSeq 500 machine.