|
| Status |
Public on Jun 30, 2022 |
| Title |
CRDU_A1 |
| Sample type |
SRA |
| |
|
| Source name |
Spinal Cord - DNGR-1 lineage traced (tdTomato)
|
| Organism |
Mus musculus |
| Characteristics |
cell line: Astrocyte
genotype: DNGR-1 +/- Tom+/-
|
| Treatment protocol |
Astrocyte differentiation: NSCs were cultured under adherent conditions for 24 hours had their NSC media removed and replaced with DMEM:F12 supplemented with BMP4 (20ng/ml). After 3 days media was replaced with fresh media containing BMP4 (20ng/ml).
|
| Growth protocol |
Growth and maintenance of adherent NSCs: Tissue culture (T25) were pre-coated for at least 1 hour with DMEM:F12 supplemented with N-2Max supplement, bFGF (20ng/ml), EGF (20ng/ml), Heparin (5ug/ml), Laminin (2ug/ml) antibiotics and glutamine. NSCs were further cultured with this media for 4 days.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
NSCs were collected after 4 days and astrocytes were collected after 6 days of culture. Cells were lysed in RLT buffer supplemented with 2-mercaptoethanol. Cell lysates were stored at -80C before RNA extraction.Total RNA was extracted with RNeasy Mini kit (Qiagen) following manufacturer protocol.
RNA (200 ng) was used to prepare cDNA libraries using the TruSeq Stranded mRNA HT Library Preparation Kit (Illumina). Libraries were normalized, pooled and then clustered using the HiSeq® 3000/4000 PE Cluster Kit (Illumina). The libraries were imaged and sequenced on an Illumina HiSeq 4000 sequencer using the HiSeq® 3000/4000 SBS kit (Illumina) at a minimum of 25 million single-end reads (76 bp) per sample.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
FRE302A16
|
| Data processing |
Trimmomatic v0.36 was used to remove the adapters and filter raw reads below 36 bases long. The filtered reads were aligned to the Mus musculus genome Ensembl GRCm38 (release 86) using RSEM v1.3.0 and STAR v2.5.2 with the “--forward-prob” parameter was set to “0.5” and all other parameters were kept as default.
Raw counts were processed using the bioconductor package DESeq2 v1.18.1 in R v3.4.3, and normalized using the DESeq method to remove the library-specific artefacts. Variance stabilizing transformation was applied to obtain normalized log2 gene expression values.
Genome_build: Ensembl GRCm38 (release 86)
Supplementary_files_format_and_content: VST log2 normalised expression values. Rows = genes; Columns = samples.
|
| |
|
| Submission date |
Feb 24, 2020 |
| Last update date |
Jun 30, 2022 |
| Contact name |
Bruno Frederico |
| E-mail(s) |
frederico.bruno@gmail.com
|
| Organization name |
Francis Crick Institute
|
| Street address |
1 Midland Road
|
| City |
London |
| ZIP/Postal code |
NW1 1AT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL21103 |
| Series (1) |
| GSE145824 |
Bulk transcriptomic comparison between in vitro propagated DNGR-1-lineage traced cells and hippocampus derived neural stem cells |
|
| Relations |
| BioSample |
SAMN14173561 |
| SRA |
SRX7793848 |
