Illumina HiSeq 1000 sequencing; Fgf and Esrrb integrate epigenetic and transcriptional networks that regulate self-renewal of Trophoblast Stem Cells
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Placenta
Cell type
Trophoblast stem cells
NA
NA
Attributes by original data submitter
Sample
ENA-CHECKLIST
ERC000011
ENA-FIRST-PUBLIC
2015-08-19T17:01:21Z
ENA-LAST-UPDATE
2018-03-09T01:01:30Z
External Id
SAMEA3391824
INSDC center name
University of Cambridge
INSDC first public
2015-08-19T17:01:21Z
INSDC last update
2018-03-09T01:01:30Z
INSDC status
public
Submitter Id
E-MTAB-3565:Dax1/Lsd1 igg control
broker name
ArrayExpress
cell type
trophoblast stem cells
common name
house mouse
sample name
E-MTAB-3565:Dax1/Lsd1 igg control
scientific_name
Mus musculus
Sequenced DNA Library
library_name
Dax1/Lsd1 igg control
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Mouse TSCs (blastocyst-derived TS EGFP line), proven to exhibit full developmental competence as the colonize all trophoblast layers in chimeras, were cultured as described previously. Transfections were performed for 6h in OptiMEM media supplemented with Fgf2 and heparin using 1% Lipofectamine 2000 (Life Technologies) on non-adherent dishes. After 24h cells were selected with 300ug ml-1 G418. Inhibitors used: 1uM LDN 193189 trihydrochloride (Axon, 1509); 2uM endo-IWR 1 (Tocris, 3532); 2uM PD0325901; 3uM CHIR99021; 50nM Gsk-Lsd1 (N-[(1R,2S)-2- phenylcyclopropyl]-4-piperidinamine, dihydrochloride), kindly provided by the Structural Genomics Consortium (http://www.thesgc.org); 10 uM SB431542; 15 uM diethylstilbestrol (DES) (Sigma). Cells (1-2x108) were fixed in 2mM DSG (80424, Sigma) in PBS at room temperature (RT) for 45min. After washing in PBS, cells were fixed again in 1% formaldehyde in TS base media at RT for 12 min. Fixation was stopped by adding glycine to a final concentration 0.125M. Cells were washed twice in PBS and resuspended in wash buffer 1 (10 mM Hepes pH 7. 5, 10 mM EDTA, 0.5 mM EGTA, 0.75% Triton X-100) and incubated at 4C for 10min. After pelleting, cells were resuspended in wash buffer 2 (10 mM Hepes pH 7.5, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) and incubated at 4C for 10min. After pelleting cells were lysed in the lysis/sonication buffer (150 mM NaCl, 25 mM Tris pH 7.5, 5 mM EDTA, 0.1% Triton, 1% SDS, 0.5% Sodium Deoxycholate) with complete protease inhibitors (Roche) on ice for 30min. Chromatin was sonicated 30sec on/30off for 25-30 cycles using the BioRuptor (Diagenode) to the average 300-bp fragments. Chromatin was diluted 1/10 with the dilution buffer (150 mM NaCl, 25 mM Tris pH 7.5, 5 mM EDTA, 1% Triton-X 100, 0.1% SDS, 0.5% Sodium Deoxycholate) containing complete protease inhibitors. Protein G magnetic Dynabeads (10004D, Invitrogen) were blocked with 1mg ml-1 BSA and tRNA at 4C for 1h and washed with buffer A (150 mM NaCl, 25 mM Tris pH 7.5, 5 mM EDTA, 1% Triton-X 100, 0.1% SDS, 0.5% Sodium Deoxycholate). Chromatin was pre-cleared with pre-blocked beads at 4C for 1h. Three hundred and fifty micrograms of chromatin and 10ug of antibody were used per each IP. Immunoprecipitation was performed over night at 4C with rotation. Pre-blocked magnetic beads were added next morning for 7-8h. Beads were washed at 4C with buffer A (150 mM NaCl, 25 mM Tris pH 7.5, 5 mM EDTA, 1% Triton-X 100, 0.1% SDS, 0.5% Sodium Deoxycholate) three times, buffer B (50 mM Tris pH8.0, 500 mM NaCl, 0.1% SDS, 0.5% Sodium Deoxycholate, 1% NP40, 1 mM EDTA), buffer C (50 mM Tris pH8.0, 250 mM LiCl, 0.5% Sodium Deoxycholate, 1% NP40, 1 mM EDTA) and rinsed with TE buffer. DNA was eluted from beads in the elution buffer (1%SDS, 0.1M NaHCO3). Samples were treated with RNAse A and Proteinase K and reverse cross-linked overnight at 65C. DNA was phenol- chloroform extracted, chloroform extracted and purified on the PCR purification columns (Qiagen) (for ChIP-seq libraries). To generate a library, DNA from 4 IPs was pooled and the NEB Next DNA Library Prep Master Mix (NEB E6040) was used according to manufacturer’s instructions. Libraries were amplified using 18 PCR cycles, purified using Agencourt AMPure XP SPRI beads (Beckman Coulter, A63881) and size-selected on an agarose gel. The DNA was extracted using a Qiaquick gel extraction kit (Qiagen) and its concentration determined using the KAPA Illumina SYBR Universal Lid Q Kit (KAPA Biosystems KK4824) and Bioanalyzer 2100 system (Agilent).