|
| 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 |
|
| Relations |
| BioSample |
SAMN07280066 |
| SRA |
SRX2959131 |
