title: GSM3992950: 54_Wapl-C6_96h_RNA_rep2; Mus musculus; RNA-Seq
library_construction_protocol: 1. ChIP-seq: All the ChIP-seq experiments, except for H3K27me3, were performed in presence of 10% HEK293T cells as an internal reference using a published protocol with small modifications (Liu et al., 2017). For chromatin preparation, the mouse embryonic stem cells were mixed with 10% HEK293T cells and cross-linked by a final concentration of 1% formaldehyde for 10 min. The cross-linking reaction was quenched using 2.0 M glycine. The cross-linked cells were then lysed and sonicated to obtain ~300 bp chromatin using Bioruptor Plus sonication device (Diagenode). For ChIP assays, antibodies were first coupled with Protein G beads (ThermoFisher Scientific), and then the sonicated chromatin were incubated overnight at 4oC with the antibody coupled Protein G beads. After over incubation, captured chromatin was washed, eluted and de-crosslinked. The released DNA fragments were purified using MiniElute PCR Purification Kit (Qiagen). The ChIP experiments were performed using the following antibodies: (1) WAPL (16370-1-AP, Proteintech), (2) CTCF (07-729, Merck Millipore), (3) RAD21 (ab154769, Abcam), (4) SOX2 (AF2018, R&D Systems), (5) OCT4 (AF1759, R&D Systems), (6) NANOG (RCAB002P-F, Cosmo Bio Co.), (7) MED1 (A300-793A, Bethyl Laboratories), (8) H3K4me3 (pAb-003-050, Diagenode), (9) H3K27ac (ab4729, Abcam), and (10) H3K27me3 (pAb-195-050, Diagenode); 2. RNA-seq: RNA was isolated following a standard TRIzol RNA isolation protocol (Ambion). The cells were lysed using 1 ml of TRIzol reagent, and 200 µl chloroform was added to the lysates. The mixture was vortexed and centrifuged at 12,000 g at 4°C for 15 min. Upper phase was homogenized with 0.5 ml of 100% isopropanol, incubated at room temperature for 10 min, and centrifuged at 4°C for 10 min. The resulted RNA pellet was washed with 75% ice-cold ethanol, dried at room temperature for 10 min, and resuspended in RNase-free water. The isolated RNA was treated with DNase using RNeasy Mini Kit (Qiagen); 3. Hi-C: Hi-C data were generated using as a previous published protocol (Rao et al., 2014) with minor modifications (Haarhuis et al., 2017). For each template, 10 million cells were harvested and crosslinked using 2% formaldehyde. Crosslinked DNA was digested in nucleus using MboI, and biotinylated nucleotides were incorporated at the restriction overhangs and joined by blunt-end ligation. The ligated DNA was enriched in a streptavidin pull-down; 4. 4C-seq was performed as previously described (Geeven et al., 2018; van de Werken et al., 2012) using a two-step PCR method for indexing described first in (Haarhuis et al., 2017). We used MboI as the first restriction enzyme and Csp6I as the second restriction fragment. 1. ChIP-seq: ChIP-seq libraries were prepared according to the protocol of KAPA HTP Library Preparation Kit (Roche); 2. RNA-seq libraries were prepared using a TruSeq Stranded RNA LT Kit (Illumina); 3. Hi-C libraries were prepared using a standard end-repair and A-tailing method; 4. 4C-seq libraries were prepared using a two-step PCR method for indexing described first in (Haarhuis et al., 2017)
attributes.tag: GEO Accession
attributes.value: GSM3992950