title: GSM8520297: Pancreas, Normal, H3K27ac rep4; Homo sapiens; ChIP-Seq
library_name: GSM8520297
library_construction_protocol: Frozen tissue samples were homogenized using the Covaris CP02 cryoPREP Automated Dry Pulverizer. Homogenized tissue was fixed rotating at room temperature for eight minutes in 1% formaldehyde (ThermoFisher catalog #28906), diluted in PBS. Fixation was quenched with 1/20th volume of 2.5M glycine. All buffers after fixation and prior to wash buffer before elution included HALT protease inhibitor (Life Technologies catalog #78438) and 1mM sodium butyrate to inhibit histone deacetylase activity. Samples were pelleted and washed twice with cold PBS. Samples were then lysed rotating at 4°C with ChIP Lysis Buffer 1 (Boston BioProducts catalog # CHP-126) for ten minutes, then with Lysis Buffer 2 (Boston BioProducts catalog # CHP-127). Samples were then washed with Sonication Buffer (CHP-133). Samples were resuspended in sonication buffer and sonicated to an average size of 100-500 bp on the Covaris E220 focused ultrasonicator, in a volume of 750 µL sonication buffer. Following sonication, samples were collected and centrifuged at 20,000xg for 10 minutes at 4°C to pellet insoluble material. The supernatant was collected and diluted in one volume of ChIP Dilution Buffer (Boston BioProducts #CHP-143), and input controls were collected. Chromatin was immunoprecipitation overnight (12-16 hours) rotating at 4°C with anti-H3K27ac antibody (Abcam, catalog #4729) conjugated to protein G Dynabeads (Life Technologies, catalog #10004D). The following day, immunoprecipitated chromatin was washed twice with low salt wash buffer (Boston BioProducts #CHP-146), once with high salt wash buffer (Boston BioProducts #CHP-147), once with LiCl wash buffer (Boston BioProducts #CHP-149), and once with Tris- based wash buffer (Boston BioProducts #CHP-148). Chromatin was eluted with ChIP Elution Buffer (Boston BioProducts #CHP-153) for one hour at 65°C with agitation at regular intervals. Eluted chromatin was reverse crosslinked for 16 hours at 65°C. Chromatin was then diluted in one volume of TE buffer, then treated with RNAse (Fisher catalog # AM2286) for 2 hours at 37°C and proteinase K (Fisher catalog # AM2548) for 1 hour at 55°C. An equal volume of phenol/chloroform (Fisher, catalog #15593031) was added to each sample, and the aqueous phase was collected in a Phase Lock Gel tube (VWR catalog #10847-802). Each sample received 1 mL ethanol, 20 µL 5M NaCl, and 30 µg GlycoBlue (Fisher Scientific, catalog # AM9516), which were then mixed by inversion and stored at −20°C overnight. The following day, the samples were centrifuged at 20,000xg for 20 minutes to precipitate the purified DNA. Pellets were washed with 800 µL cold 80% ethanol and a second centrifugation at 20,000xg for 10 minutes. Pellets were briefly air dried and resuspend in 50 µL water. Libraries were generated using the Swift Accel-NGS 2S Plus DNA library kit (Swift Biosciences catalog #21024). Samples were multiplexed six libraries per lane on an Illumina HiSeq 2500.
identifiers.namespace: GEO
identifiers.id: GSM8520297_r1