|
| Status |
Public on Jan 01, 2019 |
| Title |
intron-DARP-EGFP line scrambled siRNA replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
Human embryonic kidney
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293
reporter: endogenous intronic sequence + DARP
exogenous gene expressed: DARP-EGFP
treatment: scrambled siRNAs
ercc mix: mix1
hours post-infection: 72 hours
rin: 10
replicate: 1
|
| 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 1,000,000 cells per sample. Either ERCC mix1 or mix2 external standards (Invitrogen) were diluted 1:100 in water and 2 ul was added to each lysate.
Sequencing libraries were generated using the QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen). 200 ng RNA was used for library preparation for each sample.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
SUPT4H1_HEK293_intron.tab.gz
scramble_1
|
| 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. Post-alignment quality control reports were generated using MultiQC (Ewels et al., 2016; version 1.0).
The raw gene expression matrix was constructed from the 'forward' column of STAR's ReadsPerGene.out.tab output files 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).
GRCh38
Genome_build: tab-delimited, gzip-compressed text file with matrix of raw quantitation results for each sample.
|
| |
|
| Submission date |
Oct 24, 2018 |
| Last update date |
Jan 01, 2019 |
| Contact name |
Thomas Sandmann |
| E-mail(s) |
genomics@dnli.com, sandmann@dnli.com
|
| 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 |
GPL20301 |
| Series (1) |
| GSE121757 |
Assessing the effect of SUPT4H1 RNAi on the transcription of a repeat-containing reporter construct |
|
| Relations |
| BioSample |
SAMN10290049 |
| SRA |
SRX4934406 |
