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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