 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 30, 2019 |
| Title |
Input_+-KLFs_primed_hESC |
| Sample type |
SRA |
| |
|
| Source name |
Input_+-KLFs_primed_hESC
|
| Organism |
Homo sapiens |
| Characteristics |
source: Early Embryo
cell type: H1 hESC
culture media: mTSER
status: Overexpression in primed
chip antibody: -
|
| Treatment protocol |
Primed H1 were transduced with GFP, KLF4 or KLF17-containing lentiviral vectors and split after 48h then selected using blasticydin for the 3 following days. Naïve WIBR3dPE hESC cells in KN/2iL media were transduced with GFP or ZNF611-containing lentiviral vectors, split after 96h, then selected for a couple of passages with blasticydin on irradiated Mouse Embryonic Blasticidin-resistant (MMMbz).
|
| Growth protocol |
Conventional (primed) human ESC lines were maintained in mTSER for H1 (Male) and IPS on Matrigel, for WIBR3 (Female) on irradiated inactivated mouse embryonic fibroblast (MEF) feeders in human ESC medium (hESM) and passaged with collagenase and dispase, followed by sequential sedimentation steps in hESM to remove single cells while naïve ES cells, primed H1 and IPS were passaged by Accutase in single cells. hES media composition: DMEM/F12 supplemented with 15% fetalbovine serum, 5% KnockOut Serum Replacement, 2 mM L-glutamine, 1% nonessential amino acids, 1% penicillin-streptomycin (Lonza), 0.1 mM β-mercaptoethanol and 4 ng/ml FGF2. Naïve media composition: 500 mL of medium was generated by including: 240 mL DMEM/F12, 240 mL Neurobasal, 5 mL N2 supplement, 10 mL B27 supplement, 2 mM L-glutamine, 1% nonessential amino acids, 0.1 mM β-mercaptoethanol, 1% penicillin-streptomycin, 50 μg/ml BSA. In addition for 4i/LA: PD0325901 (1 μM), SB590885 (0.5 μM), WH4-023 (1 μM), Activin A (10 ng/mL), 20 ng/ml hLIF, Y-27632 (10 μM) and IM-12 (0-1 μM). In addition for KN/2i media: PD0325901 (1 μM), CHIR99021 (1 μM), 20 ng/ml hLIF and Doxycycline (2 µg/ml). For conversion of primed human ESC lines (WIBR3), we seeded 2-3e105 trypsinized single cells on an MEF feeder layer in hESM supplemented with ROCK inhibitor Y-27632 (10 mM). Two days later, medium was switched to 4i/LA (+/- IM12)-containing naïve hESM (Theunissen et al., 2016). WIBR3dPE cells (OCT4 GFP knock-in depleted for its primed specific Proximal Enhancer (dPE) were converted in naïve with DOX-inducible KLF2 and NANOG transgenes and maintained in 2i/L/DOX (Theunissen et al., 2014).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were cross-linked for 10 minutes at room temperature by the addition of one-tenth of the volume of 11% formaldehyde solution to the PBS followed by quenching with glycine. Cells were washed twice with PBS, then the supernatant was aspirated and the cell pellet was conserved in -80°C. Pellets were lysed, resuspended in 1mL of LB1 on ice for 10 min (50 mM HEPES-KOH pH 7.4, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10% Glycerol, 0.5% NP40, 0.25% Tx100, protease inhibitors), then after centrifugation resuspend in LB2 on ice for 10 min (10 mM Tris pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and protease inhibitors). After centrifugation, resuspend in LB3 (10 mM Tris pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% NaDOC, 0.1% SDS and protease inhibitors) for histone marks and SDS shearing buffer (10 mM Tris pH8, EDTA 1mM, SDS 0.15% and protease inhibitors) for transcription factor and sonicated (Covaris settings: 5% duty, 200 cycle, 140 PIP, 20 min), yielding genomic DNA fragments with a bulk size of 100-300bp. Coating of the beads with the specific antibody and carried out during the day at 4°C, then chromatin was added overnight at 4°C for histone marks while antibody for transcription factor is incubated with chromatin first with 1% Triton and 150mM NaCl. Subsequently, washes were performed with 2x Low Salt Wash Buffer (10 mM Tris pH 8, 1 mM EDTA, 150mM NaCl, 0.15% SDS), 1x High Salt Wash Buffer (10 mM Tris pH 8, 1 mM EDTA, 500 mM NaCl, 0.15% SDS), 1x LiCl buffer (10 mM Tris pH 8, 1 mM EDTA, 0.5 mM EGTA, 250 mM LiCl, 1% NP40, 1% NaDOC) and 1 with TE buffer. Final DNA was purified with Qiagen Elute Column. Up to 10 nanograms of ChIPed DNA or input DNA (Input) were prepared for sequencing. Library was quality checked by DNA high sensitivity chip (Agilent). Quality controlled samples were then quantified by picogreen (Qubit® 2.0 Fluorometer, Invitrogen). Cluster amplification and following sequencing steps strictly followed the Illumina standard protocol.
Libraries were ligated with Illumina adaptors
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Sequenced reads were de-multiplexed to attribute each read to a DNA sample and then aligned with bowtie2
Peak calling for ChIP-seqs were done using MACS for single-end data and MACS2 for paired-end data
Genome_build: hg19
Supplementary_files_format_and_content: A bed file containing the output of the peak called by MACS or MACS2 is provided.
|
| |
|
| Submission date |
Apr 29, 2019 |
| Last update date |
Apr 30, 2019 |
| Contact name |
Julien Duc |
| E-mail(s) |
[email protected]
|
| Organization name |
EPFL
|
| Department |
School of Life Science
|
| Lab |
LVG
|
| Street address |
Station 19 CH-1015 Lausanne
|
| City |
Lausanne |
| State/province |
VAUD |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE117395 |
Hominid-specific transposable elements and KRAB-ZFPs facilitate human embryonic genome activation and transcription in naïve hESCs |
| GSE130417 |
Hominid-specific transposable elements and KRAB-ZFPs facilitate human embryonic genome activation and transcription in naïve hESCs [ChIP-seq] |
|
| Relations |
| BioSample |
SAMN11525749 |
| SRA |
SRX5763004 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not provided for this record |
|
|
|
|
 |
