GSM9573159: BFP-transduced hiPSC-derived neurons, culture 2, DRIP-seq; Homo sapiens; OTHER
Genomic DNA was extracted from cells (roughly 1X10^6 iPSC-neurons on a 6-well plate, enzymatically detached with Accutase (Millipore) and pelleted with centrifugation for 5 mins at 1,000 G), via incubation with 20% SDS and 20 mg/mL Proteinase K overnight at 37°C. After standard phenol/chloroform extraction, 3M NaOAc pH 5.2 and 2.4 volumes of 100% ice-cold EtOH was added and DNA was precipitated via gentle spooling with a pipette tip to avoid RNA contamination that can result from centrifugation. After three gentle washes with 70% EtOH and 1-2 hours of air drying, DNA was resuspended in TE and digested with a cocktail of restriction enzymes that avoid GC-rich regions (HindIII, EcoRI, XbaI, and SSPI) overnight at 37°C. DNA was again purified via phenol/chloroform extraction and ethanol precipitation, and complete digestion was confirmed on an agarose gel.  For immunoprecipitation of R-loops, a total of 13.2 μg of digested DNA (split into 3 tubes each containing 4.4 μg DNA) was combined with binding buffer (100 mM NaPO4 pH 7.0, 1.4 M NaCl, 0.5% Triton X-100) and 10 μL of 1 mg/mL S9.6 antibody was added to each tube. Samples were incubated with rotation overnight at 4°C. The next day, magnetic protein A/G beads (Thermo Fisher Scientific, cat. no. #88803) were washed with binding buffer 3 X 10 mins and 50 μL was added to each sample tube followed by a 2 hour incubation on a rotisserie shaker at 4°C. Bead:antibody complexes were washed 3 X 10 mins with binding buffer, then 250 μL of elution buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5% SDS) and 7 μL of Proteinase K (20 mg/mL) was added to each tube and incubated at 55°C for 45 minutes with agitation to elute DNA from the S9.6 antibody. A standard phenol/chloroform extraction was performed, and DNA concentration and purity was assessed with a NanoDrop spectrophotometer. RNase A was added to samples (0.5 μL of 1 μg/μL stock) and incubated at 37°C for 1 hour. DNA was sheared with a Covaris S220 sonicator to a peak fragment size of 300 bp (duty cycle: 2%, incident power: 105 Watts, cycles per burst: 200, bath temperature: 4°C, processing time: 3 mins). NGS libraries from DRIP DNA and inputs were constructed according to the manufacturer's instructions (KAPA Hyper Prep Kit: Roche, cat. no. #KK8502 kit and single-indexed adapters from Illumina, cat. no. #IP-202-1012 + IP-202-1024. Dual-indexed libraries were constructed with Roche, cat. no. #KK8722). Library fragment sizes were restricted to between 100-500 bp with a PippinHT instrument (Sage Science). All library sizes and concentrations used for molarity calculations were confirmed on an Agilent 2100 Bioanalyzer and a Qubit 4 fluorometer (ThermoFisher Scientific) with a Qubit dsDNA HS Assay kit (ThermoFisher Scientific, cat. no. #Q32854), respectively. DRIP--sequencing was performed on an Illumina HiSeq 2500 (single-indexed libraries). All DRIP-sequencing (2x150 bp) was seqeunced to a minimum read depth of 50,000,000 reads.