title: GSM4774107: siARID1A_1uMA485_H3K27ac_rep2_CUTNRUN; Homo sapiens; OTHER
library_construction_protocol: 72 hours following siRNA transfection, 5*10^5 12Z cells were harvested and resuspended in Wash Buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1X protease inhibitor cocktail) and washed twice by centrifuge at 600 g for 3 min, and then added to the concanavalin A bead suspension and mixed on a tube rotator for 10 min at room temp. Cell/bead conjugates were resuspended in 500 mL of Antibody Buffer (Wash Buffer with 0.05% Digitonin and 2 mM EDTA) containing 5 ug of H3K27ac antibody (Active Motif, cat# 39133) or Rabbit IgG (Cell Signaling, cat#2729) and incubated in a tube nutator overnight at 4° C. The following day, cells were washed in Digitonin Buffer (Wash Buffer with 0.05% Digitonin) three times, resuspended in 250 mL of Digitonin Buffer and 12.5 mL of CUTANA pAG-MNase (EpiCypher, cat# 15-1016) was added. Cells were mixed on a nutator at room temp for 1 hr, followed by two washes in Digitonin Buffer and one wash with Low-Salt Rinse Buffer (20 mM HEPES-NaOH pH 7.5, 0.5 mM Spermidine, 1X protease inhibitor cocktail). Tubes were chilled on ice, 1 mL of Calcium Incubation Buffer (3.5 mM HEPES-NaOH pH 7.5, 10 mM CaCl2, 0.05% Digitonin) was added, and tubes were nutated at 4° C. After 2.5 min, beads were bound to magnet, supernatant was removed and 250 mL of 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 S. cerevisiae fragmented nucleosomal DNA) was added. Beads were nutated at 37° C for 30 min, followed by centrifuge at 16,000 g for 5 min at 4° C. DNA was purified using the NucleoSpin Gel and PCR Clean-up Kit (Takara, cat# 740609.50). 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, Wilmington, MA USA). Prior to PCR amplification, end-repaired and A-tailed DNA fragments were ligated to Bioo Scientific NEXTflex Adapters (Bioo Scientific, Austin, TX, USA) at a concentration of 500nM. 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., Madison, WI, USA), 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., San Diego, CA, USA) Each library was sequenced to an average depth of 75M reads. Base calling was done by Illumina RTA3 and output was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.