title: GSM2983896: TIC_HySp5; Homo sapiens; ChIP-Seq
library_construction_protocol: 24 hours after transfection, cells were collected by scraping, washed twice in pre-warmed culture medium and fixed in 1% FA solution (Sigma) for 15 minutes. The fixative was quenched by adding Glycine (125 mM) and incubation for 3 minutes. The cells were washed once in ice-cold PBS and re-suspended in 5 mL chromatin prep buffer (Active Motif), containing 0.1mM PMSF and 0.1% protease inhibitor cocktail (PIC). To release the nuclei the sample was transferred into a pre-cooled 15 mL glas Douncer and dounced with 30 strokes. After 10 minutes incubation on ice and centrifugation at 1250 g for 5 minutes at 4°C, the nuclei were re-suspended in 500 uL sonication buffer (1% SDS, 50 mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1 mM PMSF, 1% PIC). Following another 10 minutes incubation on ice, the chromatin was sonicated with a Bioblock Scientific VibraCell 75042 sonicator (Amplitude: 25%, Time: 12 minutes, 30 seconds on, 30 seconds off, 24 cycles). Note: The sonication conditions were optimized to have a fragmentation size of around 250 bp. Then 100 uL of the sonicated chromatin was added to 900 uL ChIP dilution buffer (0.1% NP-40, 0.02 M HEPES pH 7.3, 1 mM EDTA pH 8.0, 0.15 M NaCl, 1 mM PMSF, 1% PIC) and incubated with 4 ug anti-HA antibody (NB600-363, Novus Biologicals) over night at 4°C on a rotator. Next, the sample was loaded on a ChIP-IT ProteinG Agarose Column (Active Motif) and incubated for 3 hours at 4°C on a rotator. The column was washed 6 times with 1 mL buffer AM1 (Active Motif) and the DNA eluted with 180 uL of pre-warmed buffer AM4 (Active Motif). The sample was decrosslinked by adding 30 uL 10x TE buffer, 18 uL 5 M NaCl, 57 uL H2O and incubated for 5 hours at 65°C. 5 uL of RNAse A (10 ug/uL) was added and the sample incubated at 37°C for 30 minutes before adding 10 uL of Proteinase K (10 ug/uL), and further incubation for 2 hours at 55°C. The DNA was purified with the MiniElute PCR purification kit (Qiagen). For preparing the Input DNA, 5 uL sonicated chromatin was added to 5 uL 5M NaCL in 40 uL H2O, and incubated for 15 minutes at 95°C. Next the sample was incubated at 37°C for 5 minutes with 2.5 uL of RNAse A (10 ug/ul), 2.5 uL PK (10 ug/uL) was then added, and the incubation continued at 55°C for 30 minutes. 10 uL were taken for purification (MiniElute PCR purification kit from Qiagen). 5-10 ng of purified DNA were used to make libraries according to the manufacturer's protocol (Illumina). The material was sequenced with 50 bp single-end reads on the Illumine HiSeq according to the manufacturer's specifications.
attributes.tag: GEO Accession
attributes.value: GSM2983896