title: GSM2577548: CgPho4 ChIP in high Pi media w/ CgPho2; [Candida] glabrata; ChIP-Seq
library_construction_protocol: The cross-linked cell sample was resuspended in ~1 mL of ChIP lysis buffer (50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate), and mechanically lysed using a mini-Beadbeater-24 (BioSpec) with 0.5 mm Zirconia beads (BioSpec), using the following setting: 12 cycles of 30s bead beating, with 1 min on ice-water bath in between. The lysed sample was fragmented by sonication using Covaris E220 Adaptive Focus system (setting: Duty Factor: 10%; Peak Incident Power: 175; Cycles/Burst: 200; Time:150s). Afterwards, the sample was centrifuged to remove the unsoluable fraction. The supernatant containing the solubalized chromatin was subjected to immunoprecipitation. Briefly, the supernatant was incubated with either Anti-FLAG M2 magnetic beads (Sigma-Aldrich) or Anti-V5 antibody (Abcam #15828) depending on the epitope tag that was used. After 2 hours of incubation at 4C with rotation, the V5-tagged samples were additionally incubated with Dynabeads Protein-G (Thermo-Fisher Scientific #10003D). In a traditional ChIP experiment, what follows are the washing and de-crosslinking, prior to DNA extraction. In ChIP-exo, however, the washing steps are combined with part of the library construction steps. A detailed account for the protocol can be found in (Rhee and Pugh, 2012), and is also available from Active Motif ChIP-exo vA4 manual. As mentioned above, the ChIP washing step is combined with part of the library construction steps in ChIP-exo. A brief description of the steps is provided here: with the chromatin still bound to the beads, the DNA was end-polished and P7-exo adapters are ligated onto the blunt ends. The nicked DNA was repaired and then digested by lambda and RecJf exonulceases to excise DNA in a 5’ to 3’ direction, trimming up to the site of the cross-linking and selectively eliminating the P7 adapter at the 5 ́ end. Following cross-link reversal and elution from the beads, the DNA was made double-stranded by P7 primer extension and a P5- exo adapter was added to the exonuclease-treated ends. The DNA library was PCR amplified and size selected before it was subjected to high-throughput sequencing.
attributes.tag: GEO Accession
attributes.value: GSM2577548