organism: Mus musculus
description: Protocols: 30 to 40 million ESCs or TSCs were fixed in 2 % formaldehyde (Agar Scientific) for 10 minutes at room temperature. Blastocyst-derived TS-EGFP cells were cultured under routine conditions (20% fetal bovine serum, 1 mM Na-pyruvate, Pen/Strep, 50 μM 2-mercaproethanol, 25 ng/ml bFGF and 1 μg/ml heparin in RPMI1640, with 70% of the medium pre-conditioned on embryonic feeder cells). J1 ESCs were grown in feeder-free conditions using DMEM-based medium with 15% FBS and 1,000 U/ml ESGRO LIF (Millipore). Hi-C libraries were prepared as described (Comoglio et al. 2018), with the ligation performed in nuclei (Nagano et al. 2015). Biotin-marked ligation products were isolated using MyOne Streptavidin C1 Dynabeads (Life Technologies), and the bead-bound Hi-C DNA was amplified with 7 PCR amplification cycles using PE PCR 1.0 and PE PCR 2.0 primers (Illumina). Promoter Capture Hi-C was performed as described (Schoenfelder et al. 2015a), using a custom-made RNA capture bait system (Agilent Technologies) consisting of 39,021 individual biotinylated RNAs targeting the ends of 22,225 promoter-containing mouse HindIII restriction fragments. The Hi-C library DNA (500 to 750 ng) was mixed with hybridization blockers (Agilent Technologies) and denatured for 5 minutes at 95˚C, then incubated with hybridization buffer and the RNA capture bait system at 65˚C for 24 hours (in a MJ Research PTC-200 PCR machine). After the hybridization incubation, DNA/biotin-RNA was isolated using MyOne Streptavidin T1 Dynabeads (Life Technologies), following the manufacturer's instructions (SureSelect Target Enrichment, Agilent Technologies). After the final wash in wash buffer 2 (Agilent Technologies), the beads were resuspended in 300 µl NEBuffer 2 (NEB), isolated on a DynaMag-2 magnet (Life Technologies), and resuspended in a final volume of 30 µl NEBuffer 2. Hi-C libraries were prepared as described (Comoglio et al. 2018 doi/10.1101/gr.227272.117), with the ligation performed in nuclei (Nagano et al. 2015 doi.org/10.1186/s13059-015-0753-7). Biotin-marked ligation products were isolated using MyOne Streptavidin C1 Dynabeads (Life Technologies), and the bead-bound Hi-C DNA was amplified with 7 PCR amplification cycles using PE PCR 1.0 and PE PCR 2.0 primers (Illumina). Promoter Capture Hi-C was performed as described (Schoenfelder et al. 2015a doi/10.1101/gr.185272.114), using a custom-made RNA capture bait system (Agilent Technologies) consisting of 39,021 individual biotinylated RNAs targeting the ends of 22,225 promoter-containing mouse HindIII restriction fragments. The Hi-C library DNA (500 to 750 ng) was mixed with hybridization blockers (Agilent Technologies) and denatured for 5 minutes at 95˚C, then incubated with hybridization buffer and the RNA capture bait system at 65˚C for 24 hours (in a MJ Research PTC-200 PCR machine). After the hybridization incubation, DNA/biotin-RNA was isolated using MyOne Streptavidin T1 Dynabeads (Life Technologies), following the manufacturer's instructions (SureSelect Target Enrichment, Agilent Technologies). After the final wash in wash buffer 2 (Agilent Technologies), the beads were resuspended in 300 µl NEBuffer 2 (NEB), isolated on a DynaMag-2 magnet (Life Technologies), and resuspended in a final volume of 30 µl NEBuffer 2. Following library enrichment, we performed PCR (four amplification cycles using Illumina PE PCR 1.0 and PE PCR 2.0 primers), and the Promoter CHi-C libraries were purified with AMPure XP beads (Beckman Coulter).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-08-01T17:01:35Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-09T13:46:33Z
attributes.tag: External Id
attributes.value: SAMEA104678436
attributes.tag: INSDC center name
attributes.value: Clinical Pharmacology Department, William Harvey Research Institute, Queen Mary University London
attributes.tag: INSDC first public
attributes.value: 2018-08-01T17:01:35Z
attributes.tag: INSDC last update
attributes.value: 2018-03-09T13:46:33Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6585:ESC_rep1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: J1
attributes.tag: cell type
attributes.value: embryonic stem cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: inner cell mass
attributes.tag: sample name
attributes.value: E-MTAB-6585:ESC_rep1
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: 129S4/SvJae