GSM1356063: H3K27ac; Homo sapiens; ChIP-Seq
Total RNA was extracted from 1x107 Raji cells using TRIzol (Life Technologies, USA) according to the manufacturer’s instructions with some modifications to ensure higher recovery rates of small RNAs. This was achieved by addition of 10μg of linear acrylamide (Life Technologies, USA) before RNA precipitation. DNA was digested using the rigorous Turbo DNase (Ambion, USA) treatment as per manufacturer’s instructions. RNA quantity was measured on a Qubit apparatus (Life Technologies, USA) using RNA assay kit the quality was verified using RNA pico chips on a 2100 Bioanalyzer (Agilent Technologies, USA).  Before preparation of sequencing libraries, small RNAs were enriched from 10µg total RNA by using mirVana RNA Isolation kit (Life Technologies, USA) using manufacturer’s protocol for small RNA enrichment. Strand specific RNA-seq library was constructed with ScriptMiner Small RNA-seq Library Preparation Kit (Epicenter, USA) according to manufacturer’s recommended protocol. Briefly, after both 5’ and 3’ adapter ligation, resulting cDNA library was PCR amplified with 14 amplification cycles. Purified library DNA was run on a 10% TBE-PAGE gel and library DNA corresponding to transcripts between 15nt-50nt was cut from the gel and transferred into 0.5mL tubes with punctured bottoms which were in turn placed in 2mL collection tubes. Gel slices were crushed into 2mL tubes by a 2 minutes centrifugation at 14000g. For library DNA elution by soaking, 0.4ml of 0.3M NaCl was added to each tube, before a 4 hour rotation at room temperature. After removal of gel particles using 0.22μm cellulose acetate filters, 10μg of linear acrylamide (Life Technologies, USA) and 2.5 volumes (approximately 1ml) of ice-cold absolute ethanol were added. After 30 minutes incubation at -80°C, the eluted cDNA was precipitated by centrifugation at 4°C and maximum speed for 45 minutes. The pellet was washed with 1ml of cold 80% ethanol, air dried and resuspended in 20μl of water. The size-selected small RNA library DNA was quantified using a Qubit apparatus with dsDNA High Sensitivity kit (Life Technologies, USA) and verified using DNA High Sensitivity 2100 Bioanalyzer chips (Agilent Technologies, USA).
GEO Accession
GSM1356063