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| Status |
Public on Jul 08, 2020 |
| Title |
KO1_rep3 |
| Sample type |
SRA |
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| Source name |
NIH-3T3 cell culture
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| Organism |
Mus musculus |
| Characteristics |
genotype: Crls1KO1
cell line: NIH-3T3
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| Treatment protocol |
NIH-3T3 cells were seeded in 6-well plates at 60% confluency, allowed to attach, and transfected with 1.5 μg of a pD1311-AD mammalian Cas9 expression vector (ATUM) encoding a Crls1-targeting gRNA using FuGENE HD (Promega) in Opti-MEM (Invitrogen, Thermofisher). For establishing stably transfected colonies, single-cell sorting was performed on the transfected cell population 72 hours post-transfection; where individual cells were sorted into 96-well plates based on GFP fluorescence signal using a FACSAria II in PBS + 2%FBS (v/v) (BD Biosciences). Loss of Crls1 alleles was confirmed by both Sanger sequencing and Nextera sequencing of PCR amplified gDNA performed by the Australian Genomic Research Facility (AGRF).
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| Growth protocol |
NIH-3T3 cells were cultured at 37˚C in humidified 95% air/5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Life Technologies) containing: glucose (4.5 g/L), glutamine (2 mM), foetal bovine serum (10%), penicillin (100 U/ml), streptomycin sulphate (100 μg/ml), sodium pyruvate (1 mM), and uridine (50 μg/ml).
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from NIH-3T3 cells using an miRNeasy MiniKit (Qiagen), incorporating an on-column RNase-free DNA digestion, according to the manufacturer’s instructions.
Total RNA libraries were prepared according to the Illumina TruSeq protocol, using random hexamer primers for cDNA generation and the Ribo-Zero rRNA removal kit for cytoplasmic RNA deptetion.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
S6
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| Data processing |
Adaptors were trimmed from the 3' ends of sequenced reads with cutadapt (-O 1 -m 15).
Alignment was performed by Salmon v0.14.1 (-l ISR --numBootstraps 1000 --seqBias --gcBias --validateMappings --mimicBT2) using the selective alignment procedure utilising a decoy-aware transcriptome index built from the GRCm38 reference sequence and customised GENCODE vM21 gene annotation.
Differential gene expression analysis was performed with DESeq2 v1.24.0 and tximport v1.12.3, using apeglm v1.6.0 for log2 fold change shrinkage.
Genome_build: mm10
Supplementary_files_format_and_content: DESeq2 normalized gene counts for all samples in .txt format, plus DESeq2 differential expression results for each KO (1/2/3) vs WT comparison in .txt format.
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| Submission date |
Apr 06, 2020 |
| Last update date |
Jul 08, 2020 |
| Contact name |
Stefan J Siira |
| Organization name |
Harry Perkins Institute of Medical Research
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| Department |
Molecular medicine
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| Lab |
Mitochondrial medicine and biology
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| Street address |
6 Verdun St, Nedlands
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| City |
Perth |
| State/province |
Western Australia |
| ZIP/Postal code |
6009 |
| Country |
Australia |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE148178 |
Cardiolipin is required for membrane docking of mitochondrial ribosomes and protein synthesis |
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| Relations |
| BioSample |
SAMN14545584 |
| SRA |
SRX8066541 |
| 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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