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Sample GSM1697524

Query DataSets for GSM1697524

Status

Public on Apr 23, 2016

Title

E5_C1_24h

Sample type

SRA

Source name

RNA from OECs controls for 24h

Organism

Homo sapiens

Characteristics

cell type: Olfactory ensheathing cells (OECs)
infected with: none (control) for 24h

Growth protocol

O/N LB culture of Bp at 37C was collected and concentrated into PBS by centrifugation and washes in PBS

Extracted molecule

total RNA

Extraction protocol

Trizol extraction of total RNA was performed according to the manufacturer's instructions
Total RNA was subjected to two rounds of poly A+ selection and converted to cDNA. We used TruSeq library generation kits as per the manufacturer’s instructions (Illumina, San Diego, CA, USA). Library construction consisted of random fragmentation of the polyA mRNA, followed by cDNA production using random primers. The ends of the cDNA were repaired, A-tailed and adaptors ligated for indexing (up to twelve different barcodes per lane) during the sequencing runs. The cDNA libraries were quantitated using qPCR in a Roche LightCycler 480 with the Kapa Biosystems kit for library quantitation (Kapa Biosystems, Woburn, MA) prior to cluster generation. Clusters were generated to yield approximately 725K-825K clusters/mm2. Cluster density and quality was determined during the run after the first base addition parameters were assessed. We ran paired end 2X50bp sequencing runs to align the cDNA sequences to the reference genome

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Illumina bcl2fastq version 1.8.4 software was used creating the fastq files from the bcl files.
TopHat version 2.0.11 was used to align the raw RNA-Seq fastq reads to the human reference genome version 19 from the UCSC genome browser gateway using the short read aligner Bowtie version 2.1.0. TopHat was also used to analyse the mapping results to identify splice junctions between exons. See (i). Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 2009; 25:1105-11 and (ii). Langmead B, Trapnell C, Pop M, Salzberg SL. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 2009; 10:R25. and (iii). Trapnell C, Roberts A, Goff L, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 2012; 7:562-78
Cufflinks version 2.1.1 used the aligned reads from TopHat to assemble transcripts, estimate their abundances and test for differential expression and regulation. Cuffmerge, which is part of Cufflinks, merged the assembled transcripts to a reference annotation and tracked Cufflinks transcripts across multiple experiments. Finally, Cuffdiff was used to identify significant changes in transcript expression, splicing and promoter use. Genes that met certain criteria (i.e. fold change ≥ ±2.0 and q-value < 0.05) were further analyzed using the IPA tool. See (i) Trapnell C, Roberts A, Goff L, et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 2012; 7:562-78 and (ii). Trapnell C, Williams BA, Pertea G, et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 2010; 28:511-5
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited file from Cufflinks Cuffnorm that include FPKM values, normalized counts and gene symbol for each sample.

Submission date

May 27, 2015

Last update date

May 15, 2019

Contact name

Glen C Ulett

E-mail(s)

g.ulett@griffith.edu.au

Phone

61756780765

Organization name

Griffith University