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Sample GSM2685833 Query DataSets for GSM2685833
Status Public on Jul 19, 2017
Title Control RNA Rep1
Sample type SRA
Source name mesenchymal cells
Organism Gallus gallus
Characteristics breed: Lohmann Selected Leghorn
tissue: limb buds
cell type: mesenchymal cells
developmental stage: E4.5
time of culture: 5 days
condition: RCAS-BP(A)-empty
Treatment protocol Prior to seeding, mesenchymal cells were infected with RCAS-BP(A) retroviral particles carrying the CDS of connective tissue-asociated transcription factors.
Growth protocol Mesenchymal cells were isolated from limb buds of E4.5 chick embryos and were plated as 10-µL droplets at a concentration of 2.10^7 cells/mL. Cells were maintained in culture for 5 days at 37°C in DMEM/Ham’s F-12 (1:1) medium (Biochrom) supplemented with 10% FBS (Biochrom), 0.2% chicken serum (Sigma-Aldrich), 1% L-glutamine (Lonza) and 1% penicillin/streptomycin (Lonza).
Extracted molecule total RNA
Extraction protocol RNA extracts were obtained by harvesting 6 cultures with RLT buffer (Qiagen). Total RNAs were purified by using the RNeasy mini kit (Qiagen) in combination to a DNase I (Qiagen) treatment to prevent genomic DNA contamination.
RNA libraries were prepared by using the TruSeq Stranded mRNA Library Preparation kit (Illumina). Strand-specific 50-bp paired-end reads were generated by using a HiSeq 2500 sequencer (Illumina) with a mean insert size of 150 bp.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
Data processing RNA-seq strand-specific read pairs were mapped against the chicken genome galGal4 by using TopHat2 (-r 150; -N 3; --read-edit-dist 3; --library-type fr-firststrand; -i 50; -G) and a custom gene annotation model.
Alignment maps were split by strand by using SAMtools according to their FLAG field (strand plus: -f 128 -F 16, -f 80; strand minus: -f 144, -f 64 -F 16).
Fragments mapped on gene features were counted by using featureCounts (-p; -s 2; --ignoreDup; -B; -R).
Sample normalization and differential expression analysis was performed by using DESeq2 with a false-discovery rate (FDR; alpha) of 0.01.
Genome_build: galGal4
Supplementary_files_format_and_content: Tab-delimited text files containing the raw read counts for each gene for each replicate determined by featurecounts and used as input for DESeq2
Submission date Jun 26, 2017
Last update date May 15, 2019
Contact name Mickael Orgeur
Organization name Max Planck Institute for Molecular Genetics
Department Development and Disease
Street address Ihnestrasse 63-73
City Berlin
ZIP/Postal code 14195
Country Germany
Platform ID GPL19005
Series (2)
GSE100516 Genome-wide strategies identify downstream target genes of connective tissue-associated transcription factors [RNA-seq]
GSE100517 Genome-wide strategies identify downstream target genes of connective tissue-associated transcription factors
BioSample SAMN07280066
SRA SRX2959131

Supplementary file Size Download File type/resource
GSM2685833_chMM-Control_Rep1_RNA-seq.counts.txt.gz 92.5 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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