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Status |
Public on Mar 14, 2019 |
Title |
RNAi screening and timecourse Replicate 2 |
Sample type |
SRA |
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Source name |
primary Mouse Embryonic Fibroblasts transduced with tet-STEMCCA lentiviruses containing Oct4, Sox2, Klf4 and c-Myc transgenes.
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Organism |
Mus musculus |
Characteristics |
passages: Reprogrammed at passage 2 strain: BL6 genotype: Wild-type file description: This sequencing file includes all the barcoded samples corresponding to the RNAi screen and the timecourse, Replicate 2
|
Treatment protocol |
After OSKM-rtTA transduction, cells were reverse transfected with 40 nM pooled siRNAs and RNAiMAX lipofectamine. 24 hours post-transfection, cells were re-plated on gelatin-coated wells, and induced for reprogramming with doxycycline and reprogramming medium. For the timecourse the cells were not treated, only re-plated on gelatin coated wells to start reprogramming.
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Growth protocol |
Passage 1 MEFs were plated on gelatin-coated plates at a density of 10 000 cells per cm2. Next day they were incubated for 24 hours with inducible tet-OSKM (STEMCCA) and rtTA lentiviruses at an MOI of 1. Reprogramming medium was composed of DMEM, 10 % FBS, 2ug/mL doxycycline, 3uM chiron, 0.25 uM Alk5i (TGFb-inhibitor), 50 ug/mL ascorbic acid and LIF.
|
Extracted molecule |
total RNA |
Extraction protocol |
RNA was isolated with the kit Quick-RNA microprep from Zymo Research (Cat# R1051), according to the manufacturer's instructions CELseq2 library construction (T. Hashimshony et al.)
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Description |
tet-OSKM lentiviral cassette was induced with doxycycline INV_1_JPGIII003_26
|
Data processing |
Read1 and Read2 where swapped to allow compatibility with CELseq2 pipeline Reads were matched samples using the demultiplexing function of the CELseq2 pipeline Transcripts were Mapped and UMI corrected using standard settings of the CELseq2 pipeline - Reads were trimmed to 50 bases before mapping to Mus musculus genome version mm10, and hereafter matched to the gencode.vM13.annotation transcriptome Count table was generated using the standard settings of the CELseq2 pipeline Sample quality controls, filtering genes and samples, and normalization were all performed with "scater" R package version 1.3.49 Batch effect was corrected by regression with "scran" R package version 1.6.9 Genome_build: mm10 (GRCm38) Supplementary_files_format_and_content: .txt file (tab delimited) containing all counts from all samples Supplementary_files_format_and_content: .txt file (tab delimited) containing all normalized and batch effect corrected values from all samples
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Submission date |
Aug 17, 2018 |
Last update date |
Mar 14, 2019 |
Contact name |
Georgina PeƱalosa Ruiz |
E-mail(s) |
georgina@science.ru.nl
|
Organization name |
Radboud University
|
Street address |
Geert Grooteplein 30
|
City |
Nijmegen |
ZIP/Postal code |
6500HB |
Country |
Netherlands |
|
|
Platform ID |
GPL19057 |
Series (2) |
GSE118679 |
CELSeq2-profiles for siRNA screening and daily timecourse for early reprogramming to pluripotency. |
GSE118680 |
WDR5, BRCA1 and BARD1 co-regulate the DNA damage response and modulate the mesenchymal-to-epithelial transition during early reprogramming |
|
Relations |
BioSample |
SAMN09846025 |
SRA |
SRX4562188 |