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| Status |
Public on Apr 30, 2019 |
| Title |
RNA-seq_iNeurons_hESC_dCAS9-KRAB_Empty_rep1 |
| Sample type |
SRA |
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| Source name |
RNA-seq_iNeurons_hESC_dCAS9-KRAB_Empty
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| Organism |
Homo sapiens |
| Characteristics |
source: Early Embryo
cell line: IPS
status: CRISPRi in induced Neurons
culture: N2B27 + Doxycyclin
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| Treatment protocol |
WIBR3dPE in KN/2iL media or in Induced Neurons were transduced dCAS9-KRAB overexpression containing or not a guide RNA targeting LTR7YB or SVA/LTR5Hs or overexpressing. 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).
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| 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).
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| Extracted molecule |
total RNA |
| Extraction protocol |
TruSeq stranded RNA sample preparation kit (Illumina).
Truseq mRNA-seq protocol
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Reads were mapped to the human (hg19) genome using TopHat v2.0.11
Counts of reads on genes were computed using HTseq-count v0.6.1 with default parameters
Genome_build: hg19
Supplementary_files_format_and_content: .pileup files contain counts on genes.
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| Submission date |
Apr 29, 2019 |
| Last update date |
Apr 30, 2019 |
| Contact name |
Julien Duc |
| E-mail(s) |
julien.duc@epfl.ch
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| Organization name |
EPFL
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| Department |
School of Life Science
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| Lab |
LVG
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| Street address |
Station 19 CH-1015 Lausanne
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| City |
Lausanne |
| State/province |
VAUD |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE117395 |
Hominid-specific transposable elements and KRAB-ZFPs facilitate human embryonic genome activation and transcription in naïve hESCs |
| GSE130416 |
Hominid-specific transposable elements and KRAB-ZFPs facilitate human embryonic genome activation and transcription in naïve hESCs [RNA-seq] |
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| Relations |
| BioSample |
SAMN11525730 |
| SRA |
SRX5762956 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3738328_RNA-seq_iNeurons_hESC_dCAS9-KRAB_Empty_rep1.tab.gz |
11.7 Mb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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