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Status |
Public on Aug 27, 2021 |
Title |
RKO_C20_sgAAVS1_3_rep2 |
Sample type |
SRA |
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Source name |
human colon carcinoma cell line
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Organism |
Homo sapiens |
Characteristics |
tissue: Colon Carcinoma cell line: RKO treatment: AAVS1 sgRNA knockout - Day 2 method: RNA was isolated using in-house magnetic RNA isolation beads and NGS libraries were prepared with NEBNext Ultra RNA kit (NEB) according to manufacturer’s instructions and sequenced on a HiSeq2500 platform (Illumina)
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Treatment protocol |
To assess transcriptional effects of AKIRIN2, RKO cells expressing dox-inducible Cas9 were lentivirally transduced with AKIRIN2-, PSMA3- or AAVS1-targeting sgRNA and sgRNA+ cells were selected with G418. For each sgRNA, Cas9 expression was induced with dox and 2 (sgAKIRIN2 and sgAAVS1) or 3 (sgPSMA3) days post Cas9 induction cells were washed, harvested, pelleted and snap-frozen.
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Growth protocol |
RKO cells were cultured in RPMI 1640 (Gibco) and 10% fetal calf serum (FCS; Sigma). Fresh medium was added to cells 12-24h before harvest.
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Extracted molecule |
polyA RNA |
Extraction protocol |
RNA was isolated using in-house magnetic RNA isolation beads NGS libraries were prepared with NEBNext Ultra RNA kit (NEB) according to manufacturer’s instructions and sequenced on a HiSeq2500 platform (Illumina)
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Description |
RNA-Seq
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Data processing |
RNA-seq The 3' adaptors were removed using cutadapt (v1.4.2, Read1: cutadapt --match-read-wildcards -O 1 -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC) and trimmed reads with a length of less than 18bp were discarded. The trimmed reads were filtered with a contaminants database consisting of rDNA sequences (KY962518.1, NR_023363.1 and the ERCC.fa) with bowtie2 (v2.1.0 --very-sensitive-local). The recovered non matching reads were aligned to the genome/transcriptome using STAR-align (v2.4.2a, STAR --outSAMstrandField None --outFilterIntronMotifs RemoveNoncanonical --outFilterMismatchNoverLmax 0.1 --outFilterMismatchNmax 10 --outFilterScoreMinOverLread 0.30 --outFilterMatchNminOverLread 0.30 --outFilterMatchNmin 30 --chimSegmentMin 15 --quantMode TranscriptomeSAM --chimJunctionOverhangMin 15 --twopassMode Basic --outSAMtype SAM --outSAMattributes All --outReadsUnmapped Fastx intronMotif --alignIntronMax 200000 --outSJfilterIntronMaxVsReadN 10000 20000 30000 50000 --outSJfilterOverhangMin 20 12 12 12 --outFilterType BySJout --alignMatesGapMax 0 --outFilterMultimapNmax 20). The STAR-index used for alignment was generated from the human genome build NCBI GRCh38 (GCA_000001405.15) and a transcriptome GTF file from ENSEMBL build 78. Differential gene expression analysis: Read counts for genes were quantified using featureCounts v1.6.4. As a reference, we downloaded all human hg38 refSeq genes from the UCSC table browser (https://genome.ucsc.edu/cgi-bin/hgTables) on May 2, 2016. Differential gene expression analysis was performed with DESeq2 v1.22.2 and shrinking log2-foldchanges with apeglmv1.4.2. Genome_build: NCBI hg38 Supplementary_files_format_and_content: Tab-delimited table with TPMs and DESeq2 log2-foldchanges, adjusted p-values and control TPMs
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Submission date |
Sep 08, 2020 |
Last update date |
Aug 27, 2021 |
Contact name |
Tobias Neumann |
Organization name |
IMP
|
Street address |
Campus-Vienna-Biocenter 1
|
City |
Vienna |
ZIP/Postal code |
1030 |
Country |
Austria |
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Platform ID |
GPL16791 |
Series (1) |
GSE157663 |
AKIRIN2 controls the nuclear import of proteasomes in vertebrates |
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Relations |
BioSample |
SAMN16082306 |
SRA |
SRX9093639 |