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| Status |
Public on Apr 19, 2022 |
| Title |
O2_negative_control_intermediate_17days_exp2 |
| Sample type |
SRA |
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| Source name |
Fibroblast
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Negative control intermediate
length of reprogramming phase (days): 17
donor age (years): 53
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| Growth protocol |
Dermal fibroblasts from middle age donors (38-53 years old) were purchased from Lonza. For lentiviral iPSC reprogramming, fibroblasts were expanded in fibroblast medium (DMEM-F12, 10% FBS, 1X Glutamax, 1X MEM-NEAA, 1X beta-mercaptoethanol, 0.2X Penicillin/Streptomycin, 16 ng/ml FGF2) before being spinfected with tetO-GFP-hOKMS and M2rtTA lentiviruses, where 10% virus supernatant and 8 µg/ml polybrene was added to the cells before centrifugation at 1000 rpm for 60 minutes at 32°C. Reprogramming was initiated 24 hours after lentiviral transduction by introducing doxycycline (2 µg/ml) to the media. Media was changed daily throughout the experiment subsequently. On day 2 of reprogramming, cells were flow sorted for viable GFP positive cells and then cultured on gelatine coated plates. On day 7 of reprogramming, cells were replated onto irradiated mouse embryonic fibroblasts (iMEFs) and on day 8 of reprogramming, the medium was switched to hES medium (DMEM-F12, 20% KSR, 1X Glutamax, 1X MEM-NEAA, 1X beta-mercaptoethanol, 0.2X Penicillin/Streptomycin, 8 ng/ml FGF2). For transient reprogramming, cells were flow sorted at day 10, 13, 15 or 17 of reprogramming for the CD13+ SSEA4- and CD13- SSEA4+ populations. These cells were then replated on iMEFs in fibroblast medium without doxycycline and subsequently maintained like fibroblasts without iMEFs.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from cell samples with the RNeasy mini kit (Qiagen) by following the manufacturer’s instructions.
RNA-Seq libraries were prepared at the Wellcome Sanger Institute and run on a HiSeq 2500 system (Illumina) for 50 bp single-end sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Reads were trimmed with Trim Galore (version 0.6.2) and aligned to the human genome (GRCh38) with HISAT2 (version 2.1.0).
Genome_build: GRCh38
RNA-seq report: SeqMonk report with log2 RPM values for each each gene.
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| Submission date |
Jun 18, 2021 |
| Last update date |
Apr 19, 2022 |
| Contact name |
Felix Krueger |
| E-mail(s) |
fkrueger@altoslabs.com
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| Organization name |
Altos Labs
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| Department |
Bioinformatics
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| Street address |
Granta Park
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| City |
Cambridge |
| ZIP/Postal code |
CB21 6GP |
| Country |
United Kingdom |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE165177 |
Multi-omic rejuvenation of human cells by maturation phase transient reprogramming [Transient_RNAseq] |
| GSE165180 |
Multi-omic rejuvenation of human cells by maturation phase transient reprogramming |
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| Relations |
| BioSample |
SAMN19774309 |
| Supplementary data files not provided |
| Raw data are available in SRA |
| Processed data are available on Series record |
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