NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM4772985 Query DataSets for GSM4772985
Status Public on Aug 27, 2021
Title RKO_C20_sgAAVS1_3_rep2
Sample type SRA
 
Source name human colon carcinoma cell line
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)
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.
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.
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)
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Description RNA-Seq
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
 
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
 
Platform ID GPL16791
Series (1)
GSE157663 AKIRIN2 controls the nuclear import of proteasomes in vertebrates
Relations
BioSample SAMN16082306
SRA SRX9093639

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

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap