organism: Mus musculus
description: Protocols: 2. Briefly, 107 mouse ES cells were trypsinized, collected in 50ml falcon tubes in 9.3ml media and crosslinked with 1.25ml 16% formaldehyde (Thermo Scientific, Cat. No 10751395) for 10min at room temperature. Cells were quenched with 1.5ml 1M glycine, washed with PBS and lysed for 20 minutes at 4oC while rotating (lysis buffer: 10mM Tris pH 8, 10mM NaCl, 0.2% NP-40, supplemented with complete proteinase inhibitors) prior to snap freezing in 1ml lysis buffer at -80oC. Lysates were then thawed on ice, pelleted and resuspended in 650µl 1x DpnII buffer (NEB). Three 1.5ml tubes with 200µl lysate each were treated in parallel with SDS (0.28% final concentration, 1hr, 37 oC, interval shaking 500rpm, 30sec on/30sec off), quenched with trypsin (1.67% final concentration, 1hr, 37 oC, interval shaking 500rpm, 30sec on/30sec off) and subjected to a 24 hour digestion with 3x10µl DpnII (homemaid, 37 oC, interval shaking 500rpm, 30sec on/30sec off). Each chromatin aliquot was independently ligated with 8 µl T4 Ligase (240 U) in a volume of 1440µl (20 hours, 16oC). Following this, the nuclei containing ligated chromatin were pelleted to remove any non-nuclear chromatin and reverse-crosslinked. 1. Wild type E14 cells were grown on gelatin-coated plates in DMEM (Gibco, Carlsbad, CA) supplemented with 10% FBS, 10 ng/mL leukemia-inhibitory factor, penicillin/streptomycin, beta-mercaptoethanol, L-glutamine and non-essential amino-acids. 3. Ligated DNA was phenol-chloroform purified. 4. The sample was resupended in 300µl water and sonicated 13x (Pico Bioruptor, 30sec on, 30sec off) or until a fragment size of approximately 200bp was reached. Fragments were size selected using AmpureX beads (Beckman Coulter: A63881, selection ratios: 0.85x / 0.4x) and the correct size was assessed by Bioanalyzer. 2x 1-5µg of DNA were adaptor ligated and indexed using the NEBNext DNA library Prep Reagent Set (New England Biolabs: E6040S/L) and NEBNext Multiplex Oligos for Illumina Primer sets 1 (New England: E7335S/L) and 2 (New England: E7500S/L). The libraries were amplified 7x using Herculase II Fusion Polymerase kit (Agilent: 600677).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-12-30T04:03:53Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-04-03T10:02:23Z
attributes.tag: External Id
attributes.value: SAMEA5547593
attributes.tag: INSDC center name
attributes.value: Principal Investigator
attributes.tag: INSDC first public
attributes.value: 2019-12-30T04:03:53Z
attributes.tag: INSDC last update
attributes.value: 2019-04-03T10:02:23Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7840:CTCF_AID_UNT_CaptureC_rep3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: ES-E14 cell
attributes.tag: cell type
attributes.value: mouse embryonic stem cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: date of experiment
attributes.value: 11.12.2018
attributes.tag: genotype
attributes.value: E14_CTCF_AID
attributes.tag: sample name
attributes.value: E-MTAB-7840:CTCF_AID_UNT_CaptureC_rep3
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: strain
attributes.value: 1290la
attributes.tag: treatment
attributes.value: Untreated