|
| Status |
Public on Oct 31, 2020 |
| Title |
5_CT3 |
| Sample type |
SRA |
| |
|
| Source name |
Hes5.3+ cells from E5 retina
|
| Organism |
Gallus gallus |
| Characteristics |
treatment: DMSO
total rna ng: 207.9
nb of cells: 192000
|
| Treatment protocol |
Retinas were removed and incubated for 8h in culture medium (DMEM, 10% FBS, 1% penicillin/streptomycin) containing 2.5 μM RA or DMSO. Retineas were dissociated with Trypsin 0.05% for 20 minutes, and Hes5.3+ cells were sorted by FACS.
|
| Growth protocol |
Chicken eggs were kept at 37°C in an incubator. Retinas were removed and cultured in 800 μl of culture medium (DMEM, 10% FBS, 1% penicillin/streptomycin) containing either 2.5 μM RA or DMSO, and placed in a 37°C incubter with 5% CO2 for 8 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from FACS sorted cells was extracted using TRIzol reagent according to manufacturer's protocol. The SMARTer™ Ultra Low RNA kit from Clontech was used for the reverse transcription and cDNA amplification according to manufacturer’s specifications, starting with 1 ng of total RNA as input.
200 pg of cDNA were used for library preparation using the Nextera XT kit from Illumina. Library molarity and quality was assessed with the Qubit and Tapestation using a DNA High sensitivity chip (Agilent Technologies). Libraries were pooled and loaded at 2 nM for clustering on a Single-read Illumina Flow cell. Reads of 100 bases were generated using the TruSeq SBS chemistry on an Illumina HiSeq 4000 sequencer.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Mapping raw reads to reference genome was performed using STAR aligner v.2.5.3a to the UCSC Gallus gallus Galgal5 reference.
The table of counts with the number of reads mapping to each gene feature of the UCSC Gallus gallus Galgal5 reference was prepared with HTSeq v0.6p1.
The differential expression analysis was performed with the statistical analysis R/Bioconductor package edgeR v.3.18.1. Briefly, the counts were normalized according to the library size and filtered. The genes having a count above 1 count per million reads (cpm) in at least 3 samples were kept for the analysis.
The differentially expressed genes tests were done with paired data GLM (general linearized model) using a negative binomial distribution.
Genome_build: UCSC Gallus gallus Galgal5
Supplementary_files_format_and_content: Processed data files are in .csv format and contain raw counts and normalized counts for each sample.
|
| |
|
| Submission date |
Mar 05, 2020 |
| Last update date |
Oct 31, 2020 |
| Contact name |
Laurent Brodier |
| E-mail(s) |
laurent.brodier@unige.ch
|
| Organization name |
University of Geneva
|
| Department |
Molecular Biology
|
| Street address |
30 Quai Ernest-Ansermet
|
| City |
Geneva |
| State/province |
Geneva |
| ZIP/Postal code |
1211 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL23499 |
| Series (1) |
| GSE146411 |
RNA sequencing of Hes5.3+ cells from E5 chick embryonic retinas in control and retinoic acid treated conditions. |
|
| Relations |
| BioSample |
SAMN14300066 |
| SRA |
SRX7855148 |
