title: GSM5708719: CIC-WT (IDH1-WT) CIC ChIP-seq; Homo sapiens; ChIP-Seq
library_construction_protocol: For each CIC-WT (IDH1-WT) cell line replicate, two ~70–80% confluent 15-cm plates were treated with 1% formaldehyde (Sigma, St. Louis, MO, USA) in PBS for 12 min with gentle rocking, followed by treatment with 0.125 M glycine (Sigma) for 5 min. Crosslinked cells were combined and pelleted using centrifugation at 1,200 rpm for 5 min at room temperature, resuspended in 450 μl ChIP lysis buffer (50 mM Tris-HCl pH 8.0, 1% SDS, 10 mM EDTA, 1X complete EDTA-free Protease Inhibitor cocktail [PIC, Roche]), and lysed on ice for 30 min. Cells were homogenized by 6 passages through a 20-gauge needle, and nuclei pellets were obtained by centrifugation at 5,000 rpm for 10 min at 4 °C. The pellet was resuspended in 900 μl shearing buffer (10 mM Tris-HCl pH 8.0, 0.1% SDS, 1 mM EDTA, 1X EDTA-free PIC) and transferred to a 1 ml milliTUBE with AFA fiber (D-Mark Biosciences) using a 30-gauge needle. Chromatin was sonicated using a Covaris S2 sonicator using the following settings: 10% duty cycle, 6 intensity, 500 burst, 16 cycles (20 s on, 40 s off) at 4–6 °C. Insoluble debris and unfragmented chromatin were removed by centrifugation at 14,000 rpm for 12 min at 4 °C. An aliquot of chromatin was de-crosslinked overnight at 68 °C (0.2 M NaCl, 0.05 mg/ml RNase), treated with proteinase K for 30 min at 42 °C, and purified using the MinElute PCR Purification Kit (28006, Qiagen). Concentration of the purified chromatin was determined using the Qubit dsDNA high sensitivity assay (Life Technologies) and the presence of DNA fragments in the 150–300 bp size range was confirmed by separation on a 2% agarose gel. Protein A Dynabeads (Life Technologies) blocked with bovine serum albumin and salmon sperm DNA for 3 h were used to pre-clear chromatin for 2 h. For immunoprecipitation, three volumes of IP buffer (10 mM Tris-HCl pH 8.0, 1% Triton X100, 0.1% deoxycholate, 0.1% SDS, 90 mM NaCl, 2mM EDTA, 1X EDTA-free PIC) were added to 22.5 μg of chromatin, which was then incubated with 5 μg of anti-CIC antibody (Sigma, HPA044341) for 1 h at 4 °C. Blocked Protein A Dynabeads (25 μl) were then added to the chromatin and antibody and incubated overnight at 4 °C. The samples were then washed twice in low salt buffer (20 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X100, 2 mM EDTA, 150 mM NaCl) and twice in high salt buffer (same as low salt buffer except 300 mM NaCl). DNA was eluted in 100 mM sodium bicarbonate with 1% SDS and 0.05 mg/ml RNase at 68 °C for 6 h or overnight, followed by treatment with proteinase K for 30 min at 42 °C. For each IP, 20 μl of chromatin was subjected to the same treatment to serve as input controls. Eluted DNA was purified using the MinElute PCR Purification Kit. Sequencing libraries were prepared following a modified paired-end library protocol (Illumina Inc.). In brief, the DNA was subjected to end-repair and phosphorylation using T4 DNA polymerase with Klenow DNA Polymerase and T4 polynucleotide kinase, respectively, in a single reaction. 3'-A overhangs were generated using Klenow fragment (3' to 5' exo minus), and ligated to Illumina PE adapters containing 5'-T overhangs. The adapter-ligated products were purified using PCR Clean DX beads (ALINE Biosciences), then PCR-amplified with Phusion DNA Polymerase in 13 cycles using Illumina's PE primer set (Illumina). PCR product was purified using PCR Clean DX beads (ALINE Biosciences), and the DNA quality was assessed and quantified using the Caliper LabChip GX DNA High Sensitivity assay (PerkinElmer) and the Quant-iT dsDNA high sensitivity assay (ThermoFisher Scientific). Libraries were normalized and pooled and the final concentration of the pooled library was determined using a Qubit dsDNA HS Assay Kit and a Qubit fluorometer (ThermoFisher Scientific). Clusters were generated on the Illumina cluster station and sequence data were generated using an Illumina HiSeq2500 platform following the manufacturer's instructions.