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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 20, 2024 |
| Title |
shNT_2 |
| Sample type |
SRA |
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| Source name |
Primary mouse neural stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Primary mouse neural stem cells
genotype: wild type (Phf2flox/flox)
treatment: wild type (Phf2flox/flox) control, infect shNT
time: NA
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| Treatment protocol |
The shRNAs against mouse Rad21 (shRad21 #1, (#TRCN0000174832) and shRad21 #2, (#TRCN0000176084)) were purchased from Sigma. The DePinho laboratory provided non-targeting (NT) or control shRNA plasmid. Plasmid DNA was transfected into cells using Polyethylenimine (Polysciences) and the mNSC was transduced with viral particles.
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| Growth protocol |
Primary mouse neural stem cells (mNSC) were isolated from the brain cortex of E13.5 embryos, and enriched using the neurosphere formation technique. The neurospheres were then expanded by culturing on laminin (5 µg/mL, overnight, 37°C) precoated dishes, in the presence of NeuroCult Proliferation mNSC media (05702, Stem Cell Technologies) that is supplemented with fibroblast growth factor (FGF) (F0291, Sigma) and epidermal growth factor (EGF) (E4127, Sigma) at 10 and 20 ng/mL, respectively.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from cells was extracted using the RNeasy® Mini or Micro Kit (Qiagen).
The mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, a ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment.
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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 |
RNA-seq data quality was monitored via FASTQC package (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
Adapters and overrepresented sequences have been removed using cutadapt software (https://cutadapt.readthedocs.io/ en/stable/).
Further reads preprocessing was performed by trim_galore with default parameters.
Mapping of RNA-seq reads was done using bowtie2 with default parameters for RNA-seq data.
RSEM software (Li and Dewey, 2011) were used to quantify the gene-level expression.
Assembly: mm10
Supplementary files format and content: txt format; raw counts for three experimental conditions with 3 replicates each.
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| Submission date |
Feb 19, 2023 |
| Last update date |
Feb 20, 2024 |
| Contact name |
Oleg Grinchuk |
| E-mail(s) |
[email protected]
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| Organization name |
IMCB
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| Street address |
61 Biopolis Dr, Proteos
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| City |
Singapore |
| ZIP/Postal code |
138673 |
| Country |
Singapore |
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| Platform ID |
GPL21103 |
| Series (1) |
| GSE225608 |
RNA-Seq analysis of mouse neural stem cells with or wihtout Rad21 knockdown. |
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| Relations |
| BioSample |
SAMN33359141 |
| SRA |
SRX19421698 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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