title: GSM2563965: ES ZNF483-A Streptavidin ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: DNA extraction: After selection, hESCs (10cm plate, ~80% confluence) were cross-linked in 1% formaldehyde for 10min, followed with 5min incubation with 125mM glycine. Next, the cells were washed 3x with ice cold PBS and scraped in PBS/0.2mM PMSF. The cell pellet was lysed in 500μl SDS ChIP Buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.1, 150mM NaCl, freshly supplemented with protease inhibitor cocktail from Sigma). After 10min incubation on ice, the chromatin was sonicated with 60 cycles (30s on + 30s off) in Bioruptor (Diagenode). The sonicated chromatin was centrifuged (10min, 4˚C, 12000rpm) and pre-purified on 50μl pre-washed Protein A Dynabeads (Life Technologies) (1h, 4˚C, 40rpm). Pre-purified chromatin was transferred on 50μl BSA-blocked (200μg/ul BSA in ChIP SDS Buffer for 3h followed by one wash in ChIP SDS Buffer) Streptavidin Dynabeads (Life Technologies) and incubated overnight (4˚C, 40rpm). Next, the beads were collected on a magnet and washed 2x with Buffer A (2% SDS), 1x Buffer B (0.1% deoxycholate sodium, 1% Triton X-100, 1mM EDTA, 50mM Hepes pH 7.5, 500mM NaCl), followed by 1x Buffer C (250mM LiCl, 0.5% NP40, 0.5% deoxycholate sodium, 1mM EDTA, 10mM Tris-HCl pH 8.1) and 2x TE (10mM Tris-HCl pH 7.5, 1mM EDTA). All washing steps were carried out in room temperature (8min, 40rpm) and in the presence of protease inhibitor cocktail (apart from the last washing in TE). After washing, the beads were resuspended in ChIP Elution Buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1) and the chromatin was de-cross-linked (overnight,  65˚C). Next day, the eluted chromatin was separated from the beads and subjected to digestion with 5ng/µl RNase (30min at 37˚C) and 0.2µg/µl Proteinase K  (2h at 55˚C)  followed by column purification using ChIP DNA Clean and Concetrator kit (Zymo Research). DNA quantity was assessed with the picogreen dye using Qubit dsDNA HS Assay kit (Life Technologies) according to manufacturer’s protocol. RNA for RNA-seq analysis was isolated using TRIzol reagent solution according to manufacturer’s protocol. The RNA quantity and integrity number (RIN) was assessed using an Agilent BioAnalyzer 2000 (Agilent Technologies). The libraries for ChIP-seq were prepared with relevant library construction kits (NEB, Illumina, respectively) according to the manufacturer’s protocols.
attributes.tag: GEO Accession
attributes.value: GSM2563965