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: ES cells were grown at 37 °C in a 5% (v/v) CO2 on 0.1% (v/v) gelatin-coated flasks in the presence of GSK3ß and MEK inhibitors and LIF (2i/LIF medium). Complete medium comprised N2B27 (50% Dulbecco's Modified Eagle's Medium (DMEM), 50% F12 (DMEM/F12, Invitrogen), 2.5 ml of N2 and 5ml of B27 (Gibco), BSA (Gibco; final concentration 0.012%), non-essential amino acids (Gibco), glucose (final concentration 0.03 M), HEPES (final concentration 4.5 mM) and 0.1 mM beta-mercaptoethanol) was supplemented with CHIR99021, an inhibitor of GSK3ß, at a final concentration of 3µM (Trevigen) and PD0325901, a MEK inhibitor, at the final concentration of 1 µM (Trevigen) and 2 ng/ml LIF. As noted below, differentiation of ES cells from the naive state can be obtained by simple withdrawal of the 2i and LIF factors. 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: 2i_LIF
attributes.tag: Experimental Factor: cell type
attributes.value: mouse embryonic stem cell
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: input DNA