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| Status |
Public on Aug 22, 2024 |
| Title |
RKO-dox-Cas9, sgERH, nucleus, DMSO treated, biol rep 3 |
| Sample type |
SRA |
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| Source name |
Colon carcinoma
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Colon carcinoma
cell line: RKO
genotype: sgERH
treatment: DMSO
time: 5 days dox
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| Treatment protocol |
Cas9 induced knockouts were generated by 5 days of doxycycline (Sigma-Aldrich,D9891) treatment (100 ng/mL).
Inhibition of translationally coupled processes was done by treating the cells with cycloheximide (Sigma-Aldrich, C1988) at 25 μg/mL or DMSO (negative control) for 4 hours.
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| Growth protocol |
RKO cells were maintained in RPMI 1640 (Thermo Fisher Scientific, 21875) supplemented with 10% FBS (Sigma-Aldrich, F7524),L-glutamine (4 mM, Sigma-Aldrich, G7513), sodium pyruvate (1 mM, Sigma-Aldrich, S8636), and 1% penicillin/streptomycin (Sigma-Aldrich, P4333) in a humidified incubator at 37 °C and 5% CO2.
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| Extracted molecule |
nuclear RNA |
| Extraction protocol |
For subcellular fractionation, ~2x10^6 cells were gently lysed in REAP buffer (0.1% NP-40 in 1x PBS) at 4°C. Nuclei and cytoplasmic fractions were separated through centrifugation at 3k g for 60 sec at 4°C twice. RNA isolation was done with High Performance RNA Isolation kit (Vienna, VBC Molecular Tools Shop, RNAisomag) and a KingFisher Flex robot (Thermo Fisher Scientific).
Poly-A enriched RNA-seq libraries using NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs) were generated according to manufacturer's recommendations.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
X22_RKO_sgERH_NU_06_B10_DMSO_4H
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| Data processing |
RNA-seq analysis was performed using the nf-core/rnaseq pipeline (v3.13.2)
The following relevant parameters were used (.json format): { "trimmer": "fastp", "min_trimmed_reads": 2000, "remove_ribo_rna": true, "pseudo_aligner": "salmon", }
The following software versions were used (.yml format): BEDTOOLS_GENOMECOV: bedtools: 2.30.0 CUSTOM_DUMPSOFTWAREVERSIONS: python: 3.12.0 yaml: 6.0.1 CUSTOM_GETCHROMSIZES: getchromsizes: 1.16.1 DESEQ2_QC_PSEUDO: bioconductor-deseq2: 1.28.0 r-base: 4.0.3 DESEQ2_QC_STAR_SALMON: bioconductor-deseq2: 1.28.0 r-base: 4.0.3 DUPRADAR: bioconductor-dupradar: 1.28.0 r-base: 4.2.1 FASTP: fastp: 0.23.4 FASTQC_RAW: fastqc: 0.12.1 FASTQC_TRIM: fastqc: 0.12.1 FQ_SUBSAMPLE: fq: 0.9.1 (2022-02-22) GTF2BED: perl: 5.26.2 GTF_FILTER: python: 3.9.5 GUNZIP_FASTA: gunzip: '1.10' GUNZIP_GTF: gunzip: '1.10' MAKE_TRANSCRIPTS_FASTA: rsem: 1.3.1 star: 2.7.10a MULTIQC_CUSTOM_BIOTYPE: python: 3.9.5 PICARD_MARKDUPLICATES: picard: 3.0.0 QUALIMAP_RNASEQ: qualimap: 2.2.2-dev RSEQC_BAMSTAT: rseqc: 3.0.1 RSEQC_INFEREXPERIMENT: rseqc: 3.0.1 RSEQC_INNERDISTANCE: rseqc: 3.0.1 RSEQC_JUNCTIONANNOTATION: rseqc: 3.0.1 RSEQC_JUNCTIONSATURATION: rseqc: 3.0.1 RSEQC_READDISTRIBUTION: rseqc: 3.0.1 RSEQC_READDUPLICATION: rseqc: 3.0.1 SALMON_INDEX: salmon: 1.10.1 SALMON_QUANT: salmon: 1.10.1 SAMTOOLS_FLAGSTAT: samtools: '1.17' SAMTOOLS_IDXSTATS: samtools: '1.17' SAMTOOLS_INDEX: samtools: '1.17' SAMTOOLS_SORT: samtools: '1.17' SAMTOOLS_STATS: samtools: '1.17' SE_GENE: bioconductor-summarizedexperiment: 1.24.0 r-base: 4.1.1 SORTMERNA: sortmerna: 4.3.4 STAR_ALIGN: gawk: 5.1.0 samtools: 1.16.1 star: 2.7.9a STAR_GENOMEGENERATE: gawk: 5.1.0 samtools: 1.16.1 star: 2.7.9a STRINGTIE_STRINGTIE: stringtie: 2.2.1 SUBREAD_FEATURECOUNTS: subread: 2.0.1 TX2GENE: python: 3.9.5 TXIMPORT: bioconductor-tximeta: 1.12.0 r-base: 4.1.1 UCSC_BEDCLIP: ucsc: '377' UCSC_BEDGRAPHTOBIGWIG: ucsc: '445' Workflow: Nextflow: 23.04.2 nf-core/rnaseq: 3.13.2
Generation of "forward.bigWig" files were done from the ".markdup.sorted.bam" files (rnaseq NF-core pipeline) by using deeptools (version 3.5.4-foss-2022a) "bamCoverage" command with the following options: -of bigwig --filterRNAstrand forward --normalizeUsing CPM -bs 1 -p max
Generation of "reverse.bigWig" files were done from the ".markdup.sorted.bam" files (rnaseq NF-core pipeline) by using deeptools (version 3.5.4-foss-2022a) "bamCoverage" command with the following options: -of bigwig --filterRNAstrand reverse --normalizeUsing CPM -bs 1 -p max
Intron retention was analyzed with IRFinder (v. 2.0.0), and reference genome was made based on NF-core pipeline output using the following software versions: perl (5.36.0-gcccore-10.2.0), star (2.7.9a-gcc-10.2.0), samtools (1.14-gcc-10.2.0), bedtools (2.30.0-gcc-10.2.0), and minimap2 (2.18-gcccore-10.2.0)
Assembly: GRCh38 v110 (hg38)
Supplementary files format and content: tab-delimited .tsv file includes transcript count values for each sample
Supplementary files format and content: tab-delimited .tsv file includes transcript tpm values for each sample
Supplementary files format and content: tab-delimited .tsv file includes transcript length values for each sample
Supplementary files format and content: bigWig files with CPM normalized reads information for "+" strand genome track
Supplementary files format and content: bigWig files with CPM normalized reads information for "-" strand genome track
Supplementary files format and content: tab-delimited .txt files for IRFinder output information including intron IRratio values
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| Submission date |
Aug 06, 2024 |
| Last update date |
Aug 22, 2024 |
| Contact name |
Gijs A. Versteeg |
| E-mail(s) |
gijs.versteeg@univie.ac.at
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| Organization name |
Max Perutz Labs, University of Vienna
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| Department |
Department of Microbiology
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| Lab |
Versteeg
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| Street address |
Dr. -Bohrgasse 9
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| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE274074 |
ERH regulates type II interferon immune signaling through post-transcriptional regulation of JAK2 mRNA [4] |
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| Relations |
| BioSample |
SAMN43048501 |
| SRA |
SRX25617929 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8442968_22_RKO_sgERH_NU_06_B10_DMSO_4H.CPM.forward.bigWig |
146.8 Mb |
(ftp)(http) |
BIGWIG |
| GSM8442968_22_RKO_sgERH_NU_06_B10_DMSO_4H.CPM.reverse.bigWig |
146.5 Mb |
(ftp)(http) |
BIGWIG |
| GSM8442968_22_RKO_sgERH_NU_06_B10_DMSO_4H.IRFinder-IR-dir.txt.gz |
6.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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