title: GSM4801737: HI558-HA-ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: ~17,000 human IEQs were incubated with 5 mL of 1% formaldehyde (Thermo Scientific, Cat. # 28906) in PBS for 5 minutes at room temperature. To quench the formaldehyde fixative, 256 uL of 2.5 mM glycine was added, incubated for 2 minutes, and then centrifuged (500 x g) to pellet the islets. Islets were washed with 1mL each of PBS, NCP buffer 1 (10 mM HEPES pH 6.5, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100) and NCP buffer 2 (200 mM NaCl, 10 mM HEPES pH 6.5, 1 mM EDTA, 0.5 mM EGTA). Islets were then lysed in 3 mL of lysis buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.5% Empigen BB, 1% SDS, 1mM PMSF, and Roche cOmplete Mini EDTA-free Protease Inhibitor Cocktail Tablet). The islets were triturated with a 30-gauge needle ~20 times, and then sonicated using a Branson Digital Sonifier 250 with a double step microtip probe at 30% amplitude for 15 cycles (30 seconds on, 30 seconds off). The sample was then centrifuged (13000 x g, 10 minutes, 4˚C), and supernatant collected for immunoprecipitation. The supernatant was diluted with IP buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100) at a ratio 5:2, IP buffer:supernatant; 10% of the supernatant was set aside for ChIP input determination. To the IP buffer/supernatant mixture, 10 uL of anti-HA antibody (Abcam, ab9110) was used to immunoprecipitate HA-tagged Nfatc2/chromatin complexes overnight at 4˚C. The following day 100 uL Protein A/G magnetic beads (Pierce) were supplied to the immunoprecipitated mixture for 2 hours. The magnetic bead complexes were then sequentially washed with 1 mL of wash buffer #1 (2 mM EDTA, 20 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 150 mM NaCl), wash buffer #2 (2 mM EDTA, 20 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 500 mM NaCl), and wash buffer #3 (1 mM EDTA, 10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1% sodium deoxycholate, 1% NP-40 Alternative), followed by 5 washes in TE buffer. The Nfatc2/chromatin complexes were then eluted with elution buffer (200 mM NaCl, 100 mM NaHCO3, and 1% SDS) at 65˚C overnight. Eluted DNA was purified using MinElute PCR purification cleanup kit (Qiagen). Samples were prepared according the TruSeq ChIP Sample Preparation kit (Illumina Inc., San Diego, California, USA) with minor modifications. Libraries were size-selected for an average insert size of 350 bp using SPRI-based bead selection. Quality and quantity of the finished libraries were assessed using an Agilent DNA1000 chip and Qubit dsDNA HS Assay Kit, respectively. Libraries were standardized to 2 nM.