Illumina HiSeq 2000 sequencing; Trim24 ChIP-seq in embryonic stem (ES) cells grown by 2i+LIF (2iL) and serum media
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA
Attributes by original data submitter
Sample
ENA first public
2016-11-08
ENA last update
2018-03-09
ENA-CHECKLIST
ERC000011
External Id
SAMEA3497646
INSDC center alias
European Molecular Biology Laboratory
INSDC center name
European Molecular Biology Laboratory
INSDC first public
2016-11-08T17:11:23Z
INSDC last update
2018-03-09T02:13:24Z
INSDC status
public
Submitter Id
E-MTAB-3802:11Feb-Trim24-fR2
broker name
ArrayExpress
cell line
46c ES
common name
house mouse
growth condition
FBS
sample name
E-MTAB-3802:11Feb-Trim24-fR2
Sequenced DNA Library
library_name
11Feb-Trim24-fR2
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
"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. In addition 2% of each replicate was kept as an input control. After an overnight agitation at 4C, the samples were spun at 12000g for 10min. Then 90% of the solution was collected, and the volume was adjusted to 1ml with the IP buffer. Then 30 microliter Dynabeads protein A (Life technologies, 10001D) were added to the samples. Following 2 hours of rotation in the cold room, the beads were washed 6 times with ice-cold IP buffer (Tris-Cl 50mM, EDTA 5mM, NaCl 50mM, Triton X100 1%, NP40 0.5%). After that, the beads were resuspended in TE buffer plus 1% of SDS. Then the samples were boiled at 95C for 20min, and 40microg proteinase K was added for protein digestion at 55C for 30min. " 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."'