organism: Mus musculus
description: Protocols: 46c mouse ES cells were grown feeder-free on 0.2% gelatinized cell culture plates in serum media. The serum media contained DMEM high glucose (Life technologies, 11965-092) supplemented with 15% fetal bovine serum (Life technologies, 10270-106), 100 microM MEM non-essential amino acids (Life technologies, 11140-050), 1x Glutamax (Life technologies, 35050-061), 1x penicillin and streptomycin (Life technologies, 15140-122), 100 microM of 2-mercaptoethanol (Sigma, M7522), and 200ng/ml of LIF (EMBL, protein expression core facility). The cells were harvested by Stempro Accutase (Life technologies, A11105-01), and spun 5min at 200g to remove the media. Then the pellet was resuspend in 1.5% formaldehyde (Pierce, 28906) in PBS. For every 10cm dish, 10ml of 1.5% formaldehyde was used. After 15 min incubation at room temperature with occasional rotations, 125mM Glycine (Merck, 56-40-6) was added to the solution to quench cross-linking. Then the cells were washed twice with PBS, counted and aliquoted, and stored at -80C as dry cell pellets. The ChIP experiment was carried out using fixed ES cells grown in either serum or 2i condition with 2 replicates per condition. For each replicate 24 million cells were resuspended in 900 microliter TE buffer plus Complete protease inhibitor (Roche, 11873580001), and kept on ice for 5 min. Then 100 microliter Triton X-100 10% was added, and the cells were incubated another 5min on ice. Cells were washed twice by resuspending them in 10mM Tris-HCl (pH = 7.5), spinning afterward at 500g for 5min, and removing the supernatant. The nuclei obtained from 24 million cells (one replicate) were resuspended in 500 microliter of 10mM Tris-HCl (pH = 7.5), which was then sonicated in 4 Covaris microTUBEs (each tube contained 130 microliter) using Covaris S220 as follows: Duty cycle: 10%, Intensity: 5, Cycle/Burst: 200 and time: 430 seconds. Then the sheared chromatin in the 4 microTUBEs were pooled, and spun at 12000g for 10min to sediment cell debris. The supernatant was collected, and 1% of Triton X100, 0.5% of NP40, 50mM of NaCl, and 2.5 microg of Trim24 antibody (Bethyl lab, A300-815A) were added to the sheared chromatin obtained from one replicate. 2% of each replicate was kept as an input control. DNA was purified using phenol/chloroform isoamylalcohol and precipitated using glycogen and ethanol. Finally DNA was resuspended in 30??l of Tris-HCl 10mM. 'To prepare the library for Illumina sequencing, purified ChIP DNA was end-repaired by Klenow, T4 DNA polymerase and T4 polynucleotide kinase (NEB M210, M203 and M201). Then DNA fragments were subjected to A-tailing (NEB M0212), and NEBNext adapter ligation (NEB Index Primers Set 1, E7335S). Following PCR for 12 cycles, the amplicons were size-selected by mixing 50 microliter PCR products with 30 microliter of Ampure XP beads (Beckman Coulter A63880). The supernatant was collected, and again 45 microliter of AmpureXP beads was added. After 2 rounds of washing with 70% ethanol, the DNA was eluted in 50 microliter of Tris-HCl 10mM. Once again the eluted DNA was mixed with 48 microliter of AmpureXP beads, and after the washing, they were eluted by 15 microliter of Tris-HCl 10mM. Sequencing was carried out by IlluminaHiSeq 2000 according to the manufacturer''s protocols.'
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2016-11-08T17:11:23Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-09T02:13:24Z
attributes.tag: External Id
attributes.value: SAMEA3497643
attributes.tag: INSDC center name
attributes.value: European Molecular Biology Laboratory
attributes.tag: INSDC first public
attributes.value: 2016-11-08T17:11:23Z
attributes.tag: INSDC last update
attributes.value: 2018-03-09T02:13:24Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-3802:11Feb-InpFBS-R1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: 46c ES
attributes.tag: common name
attributes.value: house mouse
attributes.tag: growth condition
attributes.value: FBS
attributes.tag: sample name
attributes.value: E-MTAB-3802:11Feb-InpFBS-R1
attributes.tag: scientific_name
attributes.value: Mus musculus