title: GSM3076153: No Ab control, sequencing run 2; Homo sapiens; ChIP-Seq
library_construction_protocol: Cross-linking and harvest of cells for ChIP: To prepare material for ChIP-seq, cells were grown on 15 cm culture dishes, with two independent passages maintained simultaneously. Plates were removed from the incubator and placed at room temperature. Formaldehyde (Sigma F87750) was added directly to the culture media to a final concentration of 1%. Plates were incubated at room temperature for 10 minutes, with swirling every 2.5 minutes. Cross-linking was quenched by adding 2.5 M stock glycine to a final concentration of 0.125 M and plates were swirled to mix. Media was then aspirated and cells were washed once in 1X PBS. Eight milliliters of cold lysis buffer (5mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40 substitute, prepared fresh and sterile filtered) was then added directly to cells on the dish. Cells were then scraped, transferred to a 15 cm conical tube, and spun at 2000 rpm for 5 minutes at 4 C. The supernatant was then removed and flash frozen in liquid nitrogen at a concentration of 2e7 cells/mL for storage at -80 C. Sonication of cross-linked material for ChIP: Sonication time was determined empirically by sonicating aliquots of cross-linked material across multiple 10-cycle intervals of 30 seconds on and 30 seconds off using a Biorupter Twin with a circulating water bath in a 4 C environmental chamber. For HepG2 cells, intervals tested were from 2 10-cycle intervals, up to 6 10-cycle intervals, settling on 5 10-cycle intervals after evaluating sonication completion by agarose gel electrophoresis. Final sonication conditions are as follows: A 1 mL aliquot of cells was thawed and gently resuspended before being passed through a 20 gauge needle 20 times. Crude nuclear prep was then collected by spinning the lysate at 2000 rpm for 5 minutes at 4 C. The resulting pellet was resuspended in 300 uL RIPA buffer and then processed in a Biorupter Twin circulating bath sonicator for 50 cycles of 30 seconds on and 30 seconds off at the high setting in a 4 C environmental chamber. Following sonication, samples were spun at 16,000 rcf for 15 minutes at 4 C and the supernatant was either snap frozen and stored at -80 C or used as input for immunoprecipitation. Prior to immunoprecipitation, the total protein concentration of a sample was measured using a Bradford assay, and all sample concentrations were normalized to 1.75 mg/mL with 1X RIPA buffer and split into 1 mL aliquots. Immunoprecipitation: Sixty microliters of Protein G Sepharose beads (Sigma P3296) were used per sample. Beads were washed twice by spinning at 2500 rcf for 1 minute, removing the supernatant and adding an equal volume of 1X PBS. Beads were then resuspended in 1X PBS to 60 uL. Thirty microliters of washed beads were blocked by adding 9 uL of 0.3 mg/mL salmon sperm DNA and 12 uL 1 mg/mL BSA and incubating at 4 C for 1 hour on a rotisserie. The remaining 30 uL was added directly to a 1 mL sonicated cell aliquot and incubated for 1 hour at 4 C on a rotisserie to pre-clear. Following clearing, the sample was spun at 2500 rcf for 1 minute and 950 uL of supernatant was transferred to a new tube. Ten micrograms of antibody (Abgent AP19255B) and 30 uL of blocked bead slurry was then added and samples were incubated at 4 C overnight with rotation on a rotisserie. A no antibody control sample was also prepared from a paired aliquot by incubating overnight with 30 uL of blocked bead slurry, without addition of the primary antibody. Approximately 18 hours later, samples were washed by spinning at 2.5k rcf at 1 minute, adding 1 mL of the following buffers, incubating for 1 minute, and repeating. All washes were carried out at 4 C. Washes began with 2 1X PBS washes, 4 washes with IP wash buffer (100 mM Tris pH 7.5, 500 mM LiCl, 1% NP-40 substitute, and 1% sodium deoxycholate), and 1 wash with PBS-RIPA buffer (1X PBS, 1% NP-40 substitute, 0.5% sodium deoxycholate, and 0.1% SDS). Immunoprecipitated material was eluted off the beads by adding 200 uL elution buffer (70 mM Tris-Cl pH 8.0, 1 mM EDTA, 1.5% SDS), and incubating at 65 C for 10 minutes with vortexing every 2 minutes. Samples were then spun at 2500 rcf for 2 minutes and the supernatant was transferred to a new tube. Reversal of cross-links:To each sample (Antibody or no antibody), 13 uL of 4 M NaCl (200 mM final) was added, and samples were incubated at 65 C for 18 hours. The next morning, 20 ug of Proteinase K was added to each sample and incubated at 45 C for 60 minutes. Five volumes of Qiagen Buffer PB (QIAquick PCR purification kit) was then added to one volume of sample. This material was then processed according to the manufacturer's instructions for the QIAquick PCR purification kit, eluting twice with 30 uL volumes of pre-warmed Qiagen Buffer EB. Libraries for sequencing with Illumina high-throughput sequencing chemistry were prepared using the NEBNext Ultra II DNA Library Prep Kit (NEB E7645) with adaptors from the NEBNext Singleplex Oligos for Illumina Kit (NEB E7350) according to manufacturer's instructions. A final cycle number of 12 cycles of amplification was required during the PCR step. Libraries were multiplexed at equal concentrations based on integration under TapeStation D1000 tape traces and sequenced on an Illumina NextSeq instrument at the The Bauer Core Facility at Harvard University.
attributes.tag: GEO Accession
attributes.value: GSM3076153