title: GSM3936516: DRB qDRIP-seq +RH; Homo sapiens; OTHER
library_construction_protocol: 2x10^6 HeLa cells were lysed by SDS/Proteinase K treatment for 3 h at 37ºC. DNA was extracted by phenol-chloroform extraction using phase lock tubes and ethanol precipitated. Precipitated DNA was gently spooled and washed with 70% ethanol without centrifugation. DNA was allowed to air dry for ~20 minutes and resuspended on ice in 130 uL TE buffer. DNA was sonicated in 6x16 mm microtubes (Covaris, Woburn, MA) to a peak fragment size of 250-300 bp, performed on a Covaris machine (E220 evolution) using SonoLab 7.3 software. For each immunoprecipitation, 4.4 µg of sonicated DNA was used. For RNase H treatment, 8 ug of sonicated DNA was digested with 5 uL RNase H (New England Biolabs) in 1X RNase H digestion buffer overnight at 37ºC and purified using standard methods described above. 4 μg of DNA was bound with 10 μg of S9.6 antibody (Antibodies Incorporated, Davis, CA) in 1 X binding buffer (10 mM NaPO4 pH 7, 140 mM NaCl, 0.05% Triton X-100) overnight at 4ºC. Dynabeads Protein G beads (Thermo Fisher Scientific) were added for 2 h. The use of magnetic beads strongly increased our fold enrichment compared to agarose beads. Bound beads were washed 3 times in binding buffer and elution was performed in elution buffer (50 mM Tris pH 8, 10 mM EDTA, 0.5% SDS, Proteinase K) for 45 min at 55ºC. DNA was purified as described. DNA libraries were prepared from 3 pooled DRIPs by sample. While the DNA material from one IP (or less) is sufficient for successful library preparation, we found pooling IPs effective in minimizing technical variability due to sample handling in pilot experiments. DNA libraries were synthesized from ssDNA using the Accel-NGS 1S DNA library kit (Swift Biosciences, Ann Arbor, MI), according to the manufacturer's protocol. Using multiplexing adapters from the 1S Plus Set A Indexing Kit (Swift Biosciences), adapter-ligated DNA was amplified by PCR, then size-selected using a left/right AMPure XP size selection (Beckman Coulter). Library DNA was analyzed on a Bioanalyzer DNA HS (Agilent, Santa Clara, CA) at the Stanford Protein and Nucleic Acid Facility, quantified by qPCR using NEBNext Library Quant Kit for Illumina (New England Biolabs), then pooled and sequenced on a HiSeq 4000 (Illumina, San Diego, CA)  with paired-end 151bp reads at the Stanford Functional Genomics Facility (NIH grant S10OD018220).
attributes.tag: GEO Accession
attributes.value: GSM3936516