title: GSM1689148: SAN_ChIP-seq_ISL1; Mus musculus; ChIP-Seq
library_construction_protocol: SAN cells were first crosslinked in 2mM disuccinimidyl glutarate (Pierce) in PBS for 30 min, then subsequently in 1% formaldehyde (Sigma) in PBS for 10 min all at room temperature. The reactions were quenched by adding glycine (Sigma) to a final concentration of 125mM. The cells were immediately centrifuged (5 min, 700x g, 4°C) and washed twice with ice-cold PBS. Cells were resuspended in swelling buffer (10 mM HEPES/KOH pH7.9, 85 mM KCl, 1 mM EDTA, 0.5% IGEPAL CA-630 (Sigma), 1x protease inhibitor cocktail (Roche), 1 mM PMSF) for 5 min. Cell pellets were spun down and resuspended in 1ml RIPA buffer (10 mM Tris/HCl pH7.6, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton X-100, 1x protease inhibitor cocktail (Roche), 1 mM PMSF). Chromatin was sheared to an average DNA size of 100-400 bp by administering 10 pulses of 30s duration at 12 W power output with 60s pause on wet ice using a Misonix 3000 sonicator. The lysate was cleared by centrifugation (5min, 16000 x g, 4°C). 1% of the supernatant was kept as ChIP input. Meanwhile Protein G Dynabeads were prepared with the ISL1 antibody (39.4D5, DSHB) by incubating Dynabeads Protein G and 5 μg specific antibody in 0.5% BSA/PBS for 1 hr at 4°C on rotator, then washed twice with 0.5% BSA/PBS and brought up to the original volume with 0.1% BSA/PBS. The protein-DNA complex of interest was immunoprecipitated by rotating the supernatant with 30μl Dynabeads Protein G-coated with specific antibody overnight at 4°C (Dynabeads from Invitrogen). Beads were washed with each buffer by rotating in 1 ml buffer at 4°C for 5 min: RIPA buffer (10 mM Tris/HCl pH7.6, 1 mM EDTA, 1 mM EGTA, 0.1% SDS, 0.1% Na-Deoxycholate, 1% Triton X-100, 1x protease inhibitor  cocktail (Roche), 1 mM PMSF), LiCl buffer (0.25 M LiCl, 1% NP40, 1% NaDOC), TE plus 0.2% Triton X-100 and TE plus 50mM NaCl. Immunoprecipitated chromatin was  eluted twice with 100 μl elution buffer each (TE, 2% SDS) into fresh tubes for 30 min and 10 min, respectively, eluates were pooled, the Na+ concentration was adjusted to 300 mM with 5 M NaCl and crosslinks were reversed overnight at 65°C in a hybridization oven. The samples were sequentially incubated at 37°C for 1hr each with 0.33 mg/ml RNase A and 0.5 mg/ml proteinase K. The DNA was isolated using the ChIP DNA Clean & Concentrator (Zymo Research) according to the manufacturer’s instructions. Sequencing libraries were prepared from collected ISL1 ChIP and corresponding input DNA by blunting, A-tailing, adaptor ligation using NExtFlex barcoded adapters from Bioo Scientific. Libraries were PCR-amplified for 12-15 cycles, size selected for 225-375bp fragments by gel extraction and single-end sequenced on a Hi-Seq 2500 (Illumina) for 50 cycles.
attributes.tag: GEO Accession
attributes.value: GSM1689148