GSM5590364: EPCAM_rep1_ARID1A; Mus musculus; OTHER
Concanavalin A magnetic beads (Bangs) were washed in Binding Buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) and incubated with either anti-ARID1A (D2A8U, Cell Signaling) or Rabbit IgG (2729, Cell Signaling). Purified cells were washed in Wash Buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 5 mM sodium butyrate) then added to the conjugated antibody-bead slurry. After 10 minutes of nutating incubation at ambient temperature, Antibody Buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 5 mM sodium butyrate, 2 mM EDTA, 0.05% digitonin) was added to cell bead mixtures, and nuclear permeabilization was confirmed with Trypan blue dye. Reactions then incubated overnight at 4°C. Reactions were washed with Digitonin Buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 5 mM sodium butyrate, 0.05% digitonin) and incubated with pAG-MNase (CUTANA, EpiCypher) for one hour at room temperature, followed by an additional wash in Digitonin Buffer then Low-Salt Rinse Buffer (20 mM HEPES-NaOH pH 7.5, 0.5 mM spermidine, 5 mM sodium butyrate). Calcium Incubation Buffer (3.5 mM HEPES-NaOH pH 7.5, 10 mM CaCl2, 0.05% digitonin) was then added to activate the pAG-MNase enzyme, and the reaction was quenched after 3 minutes using EGTA-STOP buffer (170 mM NaCl, 20 mM EGTA, 0.05% digitonin, 20 µg/mL RNase A, 20 µg/mL glycogen, 0.8 pg/mL Saccharomyces cerevisiae nucleosomal DNA as spike-in). Fragments were then released into solution at 37°C followed by 5 minutes centrifugation at 16,000 x g. Eluted DNA were then purified with the NucleoSpin Gel and PCR Clean-up Kit (Takara). Libraries for CUT&RUN samples were prepared by the Van Andel Genomics Core from 0.5-1 ng of IP material, using the KAPA Hyper Prep Kit (v5.16) (Kapa Biosystems). Prior to PCR amplification, end-repaired and A-tailed DNA fragments were ligated to Bioo Scientific NEXTflex Adapters (Bioo Scientific) at a concentration of 500 nM. Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Inc.), QuantiFluor dsDNA System (Promega Corp.), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems). Individually indexed libraries were pooled and 50 bp, paired end sequencing was performed on an Illumina NovaSeq6000 sequencer using an S1, 100 cycle sequencing kit (Illumina Inc.) Each library was sequenced to an average depth of 50 million reads. Base calling was done by Illumina RTA3 and output was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.