title: Illumina HiSeq 3000 sequencing; Gain of chromatin loops marks the exit from naive pluripotency - ChIP seq for CTCF and Rad21
design: Gain of chromatin loops marks the exit from naive pluripotency - ChIP seq for CTCF and Rad21
library_construction_protocol: Sox1-GFP mouse embryonic stem cells derived from the E14tg2a line (46C) were grown at 37 °C in a 5% (v/v) CO2 incubator in GMEM (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS, Sigma), 2 ng/ml leukemia inhibitory factor (LIF, in-house), 1 mM 2-mercaptoethanol, non-essential amino acids (Gibco), L-glutamine (Gibco) and Na-pyruvate (Gibco), on gelatin-coated (0.1% v/v) dishes. Accutase (Sigma) was used for cell dissociation and diluted with cell culture medium according to the manufacturer's recommendations. Cells were passaged every second day and seeded at a density of 1.3 million cells per 10 cm in diameter culture vessel. Medium was exchanged daily. Cells were crosslinked with 1% formaldehyde for 10min at RT, glycine was added to a final concentration of 0.125M to quench formaldehyde. Cells were lysed with 1 ml of lysis buffer (10 mM Tris pH 7.5, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 1× Complete Mini EDTA free proteinase inhibitors) for 20 minutes on ice. Chromatin was sonicated to average fragment size of 400bp. Chromatin extracts corresponding to 10M of cells (90µg of DNA) were precleared with 40µl of Protein A dynabeads. The sample was incubated at 4°C with overhead mixing for 1 hour. 40µl of protein A Dynabeads was washed with PBS and incubated with anti-CTCF (Milipore, 07-729, 5µl) or anti-Rad21 (Abcam, ab-992, 5µg) at RT with overhead mixing (40µl of beads per 5µl of anti-CTCF or 5µg of anti Rad21). 500µl of pre-cleared chromatin was mixed with the antibody-coupled beads and incubated overnight at 4°C with overhead mixing. Next, beads were washed for 10 minutes at 4°C with overhead mixing with lysis buffer (2x), lysis buffer containing 0.3M NaCl (2x), LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate, 2x), TE (pH 8.0) plus 0.2% Triton X-100, and TE (pH 8.0). Beads were resuspended in 100µl of TE (pH 8) and incubated at 65°C for 14 hours, 1µl of RNAseA was added and the sample incubated for 1 hour at 37°C. Next,10µl of Proteinase K was added and the sample incubated at 55°C for 2 hours. Beads were separated from the solution using a magnet. 110µl of the supernatant was mixed with 200µl of Ampure Beads (Beckman Coulter) and DNA was extracted following manufacturer's suggestions. The Library was prepared in Ovation SP Ultralow library system (Nugen) using 2ng of ChIP material.
attributes.tag: Experimental Factor: growth condition
attributes.value: FBS
attributes.tag: Experimental Factor: cell type
attributes.value: mouse embryonic stem cell
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: CTCF