|
| Status |
Public on Mar 06, 2014 |
| Title |
ARN_cl1_RA_12h_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
TAF4-/- immortalized mouse embryonic fibroblasts.
|
| Organism |
Mus musculus |
| Characteristics |
genotype: TAF4-/-
cell type: immortalized mouse embryonic fibroblasts
treatment: retinoic acid
time: 12 h
genetic background: C57BL/6
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Sequencing libraries were prepared from 1 µg of total RNA using reagents from the DpnII Digital Gene Expression Tag Profiling kit (Part # 1003803 Rev. B protocol, Illumina). mRNA was captured on magnetic oligo(dT) beads and reverse transcribed into single-strand cDNA and double-stranded cDNA was then synthesized. The cDNA attached to the beads was digested using the restriction enzyme DpnII. A DpnII adapter was ligated to the DpnII cleavage sites. The adapter-ligated cDNA was then digested with MmeI to release 17bp fragments of cDNA from the magnetic bead. The fragments were dephosphorylated and purified by phenol-chloroform. A second adapter was ligated at the MmeI cleavage sites. The adapter-ligated cDNA fragments were amplified by PCR (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 15 sec at 72°C] x 15 cycles; 10 min at 72°C) and the PCR products were purified on a 6% TBE PAGE gel. The 85 bp PCR products were excised from the gel and eluted overnight, followed by ethanol precipitation. DNA libraries were checked for quality and quantified using 2100 Bioanalyzer (Agilent).The libraries were loaded in the flowcell at 8pM concentration and clusters were generated following Illumina’s instructions. Libraries were sequenced on the Illumina Genome Analyzer II as single read 18 bases reads, following the manufacturer's protocols.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Illumina Pipeline v1.4 software was used for image analysis and basecalling.
The 16-base long reads (excluding the 4-base DpnII recognition site) were aligned to DpnII tag tables generated by Stowers Institute http://research.stowers-institute.org/microarray/tag_tables/index.html using Eland.
Genome_build: mm9
Supplementary_files_format_and_content: The NormalizedTagCountPerGenes.tsv file is a tab separated text file containing the normalized number of reads for each Ensembl Gene (Annotations from Ensembl Core v53_37f) .
|
| |
|
| Submission date |
Dec 06, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Igor Martianov |
| E-mail(s) |
martiano@igbmc.fr
|
| Phone |
0033388653440
|
| Organization name |
IGBMC
|
| Street address |
1 rue Laurent Fries
|
| City |
Illkirch |
| ZIP/Postal code |
67400 |
| Country |
France |
| |
|
| Platform ID |
GPL11002 |
| Series (1) |
| GSE53097 |
Retinoic acid induced changes in gene expression in TAF4 knock out fibroblasts |
|
| Relations |
| BioSample |
SAMN02438048 |
| SRA |
SRX388718 |
