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Sample GSM3223415 Query DataSets for GSM3223415
Status Public on Jan 01, 2019
Title S622: DARP-EGFP line 2, scrambled siRNAs, replicate 2
Sample type SRA
Source name Human embryonic kidney
Organism Homo sapiens
Characteristics cell line: HEK293
tissue: Human embryonic kidney
exogenous gene expressed: DARP-EGFP
treatment: scrambled siRNAs
ercc mix: mix2
hours post-infection: 72 hours
rin: 10
replicate: 2
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.
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
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).
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
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:
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.
Submission date Jun 26, 2018
Last update date Jan 01, 2019
Contact name Thomas Sandmann
Organization name Denali Therapeutics
Street address 161 Oyster Point Blvd
City South San Francisco
State/province California
ZIP/Postal code 94080
Country USA
Platform ID GPL24676
Series (1)
GSE116267 Global effects of SUPT4H1 RNAi on gene expression of HEK293 cells
BioSample SAMN09487613

Supplementary data files not provided
Raw data are available in SRA
Processed data are available on Series record

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