|
| Status |
Public on Jun 18, 2020 |
| Title |
Wild-type replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
Neural stem cell
|
| Organism |
Mus musculus |
| Characteristics |
strain: FVB/N
genotype: Smchd1 Wild-type
cell type: Neural stem cell
batch: 1 and 2
|
| Treatment protocol |
N/A
|
| Growth protocol |
NSCs were derived as previously described (Chen et al., 2015). Cells were grown in NeuroCult Stem Cell medium (StemCell Technologies #05702) with cytokines: ◦ 45ml NeuroCult NSC Basal Medium (Mouse) (StemCell Technologies #05700) ◦ 5ml NeuroCult Proliferation Supplement (Mouse) (StemCell Technologies #05701) ◦ 4μl (0.25mg/ml) rh EGF (StemCell Technologies #02633) ◦ 4μl (0.25mg/ml) rh bFGF (StemCell Technologies #02634).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
We extracted total RNA with Trizol and purified polyA RNA with the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490).
We prepared direct cDNA nanopore libraries with the DCS108 kit (batch 1) and DCS109 kit (batch 2) and barcoded libraries with the native barcoding kit (NB103 and 114). Samples were sequenced on two FLO-PRO002 PromethION flow cells.
Single-end
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
PromethION |
| |
|
| Data processing |
PromethION software version 18.02.6 (batch 1) and 19.01.6 (batch 2).
Base calling and quality scoring: Guppy version 4.0.11
Trimming and de-multiplexing: Guppy version 4.0.11
Sequenced reads were mapped using minimap2 version 2.17 with arguments -ax splice -uf -k14 --junc-bed.
Gene-level counts were obtained using the featureCounts function (Liao et al. Bioinformatics, 2014) from the Rsubread package (version 1.34.4) using GENCODE vM23 annotation.
Data were TMM normalized (Robinson and Oshlack, Genome Biology 2010) in the edgeR package (version 3.26.5, Robinson et al. Bioinformatics 2010) and transformed to log2 counts per million.
Gene-wise linear models (Smyth, SAGMB 2004) with observational level weights (Law et al. Genome Biology 2014) were fitted to summarise the values from replicates samples using functionality from the limma package (version 3.40.6, Ritchie et al. NAR, 2015).
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: *.tsv: tab-delimited text files with columns representing Ensembl Gene ID, total gene length (total base count of all exons) and the number of reads mapping to that gene.
|
| |
|
| Submission date |
Jun 04, 2020 |
| Last update date |
Nov 05, 2020 |
| Contact name |
Xueyi Dong |
| E-mail(s) |
dong.x@wehi.edu.au
|
| Organization name |
Walter and Eliza Hall Institute of Medical Research
|
| Street address |
1G ROYAL PARADE
|
| City |
PARKVILLE |
| State/province |
VIC |
| ZIP/Postal code |
3052 |
| Country |
Australia |
| |
|
| Platform ID |
GPL26624 |
| Series (1) |
| GSE151841 |
Long-read Nanopore sequencing of mouse neural stem cells |
|
| Relations |
| BioSample |
SAMN15104963 |
| SRA |
SRX8473461 |
