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Sample GSM3587857 Query DataSets for GSM3587857
Status Public on Jan 24, 2022
Title Seq22@2@S9
Sample type SRA
 
Source name Tunica albuginea
Organism Homo sapiens
Characteristics diagnosis: Control
tissue: Tunica albuginea
Extracted molecule total RNA
Extraction protocol RNA extraction: Tripure (Trizol reagents)
RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Only high quality RNA (RIN>7) was used for further processing. Per sample, an amount of 250ng of total RNA was used as input. Using the Illumina TruSeq® Stranded Total RNA Sample Prep Kit with Ribo-Zero Gold (protocol version “April 2013”) rRNA is depleted from the total RNA samples using Ribo-Zero ribosomal RNA reduction chemistry. Subsequently, RNA was purified and fragmented and converted into first strand cDNA in a reverse transcription reaction using random primers. Next, double-stranded cDNA was generated in a second strand cDNA synthesis reaction using DNA PolymeraseI and RNAse H. The cDNA fragments were extended with a single ‘A’ base to the 3’ ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichment PCR was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. Sequence-libraries of each sample were equimolarly pooled and sequenced on a NextSeq500 flow-cell with a High Output 75bp kit (Single Read - 1.3pM Pool + 1.86% PhiX v3) at the VIB Nucleomics core (www.nucleomics.be).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
Data processing Preprocessing: Low quality ends and adapter sequences were trimmed off from the Illumina reads with FastX 0.0.14 and Cutadapt 1.15 (HannonLab, 2010; Martin, 2011). Subsequently, small reads (length < 35 bp), polyA-reads (more than 90 % of the bases equal A), ambiguous reads (containing N), low-quality reads (more than 50 % of the bases < Q25) and artifact reads (all but three bases in the read equal one base type) were filtered using using FastX 0.0.14 and ShortRead 1.36.0 (Morgan et al, 2009). With Bowtie2 2.3.3.1 we identified and removed reads that align to phix_illumina (Langmead & Salzberg, 2012).
Mapping: The preprocessed reads were aligned with STAR aligner v2.5.2b to the reference genome of Homo sapiens (GRCh38) (AD et al, 2013). Default STAR aligner parameter settings were used, except for ‘--outSAMprimaryFlag OneBestScore --twopassMode Basic --alignIntronMin 50 --alignIntronMax 500000 --outSAMtype BAM SortedByCoordinate’. Using Samtools 1.5, reads with a mapping quality smaller than 20 were removed from the alignments (Li et al, 2009).
Counting: The number of reads in the alignments that overlap with gene features were counted with featureCounts 1.5.3 (Liao et al, 2014). Following parameters were chosen: -Q 0 -s 2 -t exon -g gene_id. We removed genes for which all samples had less than 1 count-per-million. Raw counts were further corrected within samples for GC-content and between samples using full quantile normalization, as implemented in the EDASeq package from Bioconductor (Risso et al, 2011).
Genome_build: Homo sapiens (GRCh38)
Supplementary_files_format_and_content: Raw counts are in tab-delimited text file.
Supplementary_files_format_and_content: Statistical Table resulting from the EdgeR analysis in tab-delimited text file.
 
Submission date Feb 01, 2019
Last update date Jan 24, 2022
Contact name Rekin's Janky
E-mail(s) Nucleomics.Bioinformatics@vib.be
Organization name VIB
Department Nucleomics Core
Street address Herestraat 49 Box 816
City Leuven
ZIP/Postal code B-3000
Country Belgium
 
Platform ID GPL18573
Series (1)
GSE126005 Deep sequencing reveals an acute inflammatory signature in chronic Peyronie’s disease
Relations
BioSample SAMN10861335
SRA SRX5323943

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

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