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| Status |
Public on Jan 01, 2019 |
| Title |
S458: EGFP line, scrambled siRNAs, replicate 2 |
| Sample type |
SRA |
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| Source name |
Human embryonic kidney
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
tissue: Human embryonic kidney
exogenous gene expressed: EGFP
treatment: scrambled siRNAs
ercc mix: mix2
hours post-infection: 72 hours
rin: 9.8
replicate: 2
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| Treatment protocol |
Cells were transfected at 50% confluency in 6-well plates by combining either SUPT4H1 siRNA or scramble siRNA with 9 ul of RNAi max transfection reagent (Invitrogen) at a final concentration of 100 nM in a total volumne of 1.3 ml.
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| Growth protocol |
HEK293 CRL-1573 (ATCC) cells were cultured in DMEM, 10% FBS, 100 units/ml penicillin and streptomycin, 37 deg.C, 5% carbon dioxide, 100% humidity
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| Extracted molecule |
total RNA |
| Extraction protocol |
72 hours post transfection, total RNA was extracted with the RNEasy (Qiagen) kit from 750,000 cells per sample. Either ERCC mix1 or mix2 external standards (Invitrogen) were diluted 1:100 in water and 1 ul was added to each lysate.
Sequencing libraries were generated by SeqMatic (Fremont, CA). rRNA was depleted with the Ribo-Zero Gold rRNA Removal Kit (Illumina) and sequencing libraries were prepared with the TruSeq Stranded Total RNA kit (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Sequencing adapters were trimmed using skewer (Jiang et al., 2014; version 0.2.2) with default parameters. Quality control of the trimmed reads was performed using FastQC (version 0.11.5: www.bioinformatics.babraham.ac.uk/projects/fastqc).
Reads were aligned to a composite index of the human genome (version GRCh38_p10) and sequences of the 92 ERCC spike-in transcripts: A STAR index (Dobin et al., 2013; version 2.5.3a) was built with the --sjdbOverhang=50 argument. Splice junctions from Gencode gene models (release 27) were provided via the --sjdbGTFfile argument. STAR alignments were generated with the following parameters: --outFilterType BySJout --quantMode TranscriptomeSAM --outFilterIntronMotifs RemoveNoncanonicalUnannotated --outSAMstrandField intronMotif --outSAMattributes NH HI AS nM MD XS --outSAMunmapped Within
Transcript abundance was quantified based on the STAR alignments using salmon (Patro et al., 2017; version 0.9.1) with the following parameters: --seqBias --gcBias --biasSpeedSamp 10 with Gencode gene models (version 27). Post-alignment quality control reports were generated using MultiQC (Ewels et al., 2016; version 1.0).
Transcript abundances were imported with the tximport Bioconductor package (Soneson et al., 2015; version 1.6.0) and aggregated to gene level counts using the "scaledTPM" option using R (version 3.4.3). Gene symbols and Entrez gene identifiers were mapped using Ensembl (version 86) via the biomaRt R package (Durinck et al, 2009; version 2.34.0).
Genome_build: GRCh38
Supplementary_files_format_and_content: tab-delimited, gzip-compressed text file with matrix of raw quantitation results for each sample.
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| Submission date |
Jun 26, 2018 |
| Last update date |
Jan 01, 2019 |
| Contact name |
Thomas Sandmann |
| E-mail(s) |
[email protected], [email protected]
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| Organization name |
Denali Therapeutics
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| Street address |
161 Oyster Point Blvd
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| City |
South San Francisco |
| State/province |
California |
| ZIP/Postal code |
94080 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE116267 |
Global effects of SUPT4H1 RNAi on gene expression of HEK293 cells |
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| Relations |
| BioSample |
SAMN09487615 |
| Supplementary data files not provided |
| Raw data are available in SRA |
| Processed data are available on Series record |
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