title: GSM2639664: APC ChIP-seq replicate 1; Homo sapiens; ChIP-Seq
library_construction_protocol: Two independent biological replicates were performed.  6 x 106 HCT-116 cells were initially seeded in each 150-mm dish.  Cells were crosslinked after 24 hrs. with 1% formaldehyde for 10 minutes before quenching by 0.125 M glycine.  Cells were washed and scraped into Dulbecco’s PBS, pelleted and resuspended in cold 10 mM HEPES pH 7.5, 10 mM EDTA, 0.5 mM EGTA, 0.75% Triton X-100, 1 mM PMSF with mammalian protease inhibitor cocktail (Sigma-Aldrich) and incubated 10 minutes at 4oC with rotation.  Cells were again pelleted and resuspended in cold 10 mM HEPES pH 7.5, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1 mM PMSF and protease inhibitors and rotated 10 minutes at 4oC.  Nuclei were pelleted and resuspended in 1 mL of cold 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.5% Sodium Deoxycholate, 1 mM PMSF with protease inhibitors.  Probe-based sonication at 4oC was performed using 30 pulses of 10-seconds each at 35% amplitude.  Fragment size was reduced by dividing the resulting supernatant into 300 μL aliquots and mixing each with 100 μL of 2 M sucrose, 320 mM KCl, 80 mM HEPES pH 7.5, 14 mM ATP, 24 mM CaCl2 and 4 µL of 50 U/µL micrococcal nuclease (Affymetrix, Inc.).  Digest was incubated 15 minutes at room temperature and quenched by mixing on ice with 45 µL of 200 mM EDTA, 20 mM EGTA.  An equal volume of 16.7 mM Tris-HCl pH 8, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, 1 mM PMSF, with protease inhibitors was added to each sample to dilute it.  Pre-clearing of input was performed using 2-hour incubation with 5 μg rabbit IgG antibody and 40 μL of pre-equilibrated Protein G Dynabead slurry (Thermo Fisher Scientific) at 4oC with rotation.  100 μL of the resulting supernatant was saved as “pre-cleared input”.  The remainder was used for anti-APC ChIP in combination with 10 μg of anti-APC antibody (a polyclonal antibody recognizing the C-terminal 50 amino acids of APC, catalog #A300-981A, Bethyl Laboratories, Inc.).  Immunoprecipitations were rotated overnight at 4oC and mixed with 40 μL of pre-equilibrated Protein G Dynabead slurry and rotated 3 hours at 4oC.  Beads were collected by magnet and washed twice with cold 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF, and protease inhibitors, twice with cold 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF, and protease inhibitors, twice with cold 50 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 1 mM PMSF, and protease inhibitors, and twice with cold 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM PMSF, and protease inhibitors.  All washes were accompanied by 5 minute rotation at 4oC.  Elution was performed by the addition of 210 μL room temperature Elution Buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, 1% SDS) to the washed beads, and 45 minute incubation at 65oC with occasional gentle resuspension of beads.  Supernatant was isolated and crosslinks were reversed by mixing with 16 μL of 5M NaCl and incubation at 65oC for 12 hrs.  210 μL TE was added to ChIP reactions, while 300 μL TE was added tothe pre-cleared input samples.  8 μL of 10 mg/mL RNase A was mixed with each sample and incubated 2 hrs at 37oC.  4 μL of 25 mg/mL Proteinase K was mixed with each sample and followed by 2 hours incubation at 55oC.  Samples were extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1), once with chloroform, and mixed with 1/10th volume of sodium acetate pH 5.2 and 60 μg glycogen for ethanol precipitation.  Yields from multiple parallel α-APC ChIP reactions were pooled to obtain the 10 ng required for ChIP-seq library preparation.  The PCR purification kit (QIAGEN) was used to clean up reactions prior to library preparation. Library preparation for ChIP-seq was performed with the NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina, using adaptors AD005 and AD019 (New England Biolabs) for the input and ChIP libraries, respectively.  Size selection was performed by E-gel (Life Technologies, Inc.) to obtain fragments ranging in size from 300-400 bp.  Next-generation sequencing (50 bp, single-end) was performed by the OSU Genomics Shared Resource using a HiSeq 2500 instrument (Illumina, Inc.).
attributes.tag: GEO Accession
attributes.value: GSM2639664