GSM4455966: C2C12 Cells IgG ChIP-seq 3; Mus musculus; ChIP-Seq
Twenty million cells per time point were used for each ChIP sample. Cells were fixed for 10 min using 1% formaldehyde in DMEM. Cell fixation was quenched by removing the fixation solution, rinsing the plates with PBS, and then pouring a solution of 0.125 M glycine in PBS onto the cells and incubating for 5 min. Subsequently, plates were washed 2× with PBS and then cells were scraped from the plates, pelleted, and stored at -80 °C until used.  Given that SATB2 is a nuclear matrix attachment protein, it required an alternative protocol for chromatin shearing than standard preparations. First, a commercial hypotonic solution (Active Motif) was used to lyse cellular membranes but retain intact nuclear membranes. We subsequently performed additional lysis using a hand held glass Dounce homogenizer. After centrifugation, the supernatant was discarded and the pelleted nuclei were treated with Active Motif's Pro-enzymatic Digestion Nuclear Extraction Solution. After incubation at 37 °C for 5 min, we supplemented the reaction with 1 U micrococcal nuclease (New England Biolabs) and allowed it to incubate for 10 min. We then supplemented the reaction with SDS to a final volume 2% SDS-chromatin solution. We proceeded to the second stage of shearing using sonication via a Covaris M220 Focused-Ultrasonicator instrument, setting the parameters to produce sheared DNA of 200 bp. After sonication, we performed a final centrifugation at 16 000 × g at 4 °C for 20 min and collected the supernatant containing the sheared chromatin.   We performed immunoprecipitations with 5 μg 1°Ab (Target: SATB2, Abcam, positive control: RNA pol II, Active Motif, negative control: mouse IgG, Santa Cruz) using magnetic beads (Active Motif) diluted in commercial ChIP-buffer (Active Motif) overnight at 4 °C. Captured chromatin was isolated using magnetic stands to pulldown the beads. Bound DNA was subsequently eluted with commercial elution, reverse cross-linking, protein digestion, and RNA digestion solutions (Active Motif). The resulting samples of DNA were further purified using phenol:chloroform extraction procedures. Final DNA was quantified using a NanoDrop spectrophotometer, with a 25 μL final   volume having concentrations of 75–100 ng/μL. For genome-wide analysis, immunoprecipitated DNA was amplified and 75 bp single read sequencing was performed on an Illumina HiSeq 2500 at the Next-Generation  Sequencing Facility at The Centre for Applied Genomics in The Hospital for Sick Children (Toronto, Ontario, Canada).