|
| Status |
Public on Apr 24, 2014 |
| Title |
RNA_UO_replicate-1 |
| Sample type |
SRA |
| |
|
| Source name |
NIH3T3_RNA_UO
|
| Organism |
Mus musculus |
| Characteristics |
cell line: NIH3T3 fibroblasts
genotype: normal
chip antibody: none
|
| Treatment protocol |
Cells were starved overnight in 0.3%FCS. Where indicated, cells were stimulated for 30 minutes with 15% fetal calf serum (FCS) or with cytochalasin D (Calbiochem, CD, 2 μM). When indicated cells were pre-traeted with latrunculin B (Calbiochem, LatB, 0.3 μM) and/or U0126 (Promega, 10 μM). Pre-treatments were for 30 minutes prior stimulation.
|
| Growth protocol |
NIH-3t3 were cultured in Dulbecco modified Eagles medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100 IU/ml penicillin, and 100 μ/ml streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Chip-Seq: DNA samples were end repaired, poly-A tailed and Illumina single end adapters were ligated following the standard Illumina protocol with minor adjustments. Agencourt AMPure XP beads at 0.8x ratio were used to size select out adapter dimers after adapter ligation. The Illumina kit Phusion enzyme was replaced by Kapa HiFi HotStart ready mix. Post PCR, AMPure XP beads were used at a 1:1 ratio to maintain size integrity and to allow use of the Invitrogen SizeSelect E-gel system. Samples were finally purified with QAIquick gel extraction kit and quality controlled on the DNA 1000 BioAnalyser 2100 chip before clustering and subsequent 36bp single end sequencing on the GAIIx analyser or on a Hi-seq 2000/1000.
RNA-Seq: Total RNA was extracted using Genelute (Sigma). RNA samples were quality controlled (QC) using the 6000 Nano RNA Chip on the BioAnalyser 2100 (Agilent). Libraries were prepared using the Directional mRNA-Seq Library Prep. v1.0. Pre-Release Protocol from Illumina with minor adjustments. To minimise the ribosomal content of the samples a DSN treatment of the cDNA RNA-seq libraries was used (Evrogen JSC).The Illumina kit Phusion enzyme was replaced by Kapa HiFi HotStart ready mix which reduced the overall volume of the PCR and the ratio for the Agencourt AMPure XP beads was adjusted accordingly. The standard PCR cycling was also changed to match the concentration of the total RNA from the initial QC. After passing the final QC, the libraries were subjected to cluster formation and then 72bp single end sequencing on the GAIIx analyser.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
Sample 65
|
| Data processing |
ChipSeq MRTFA MRTFB ELK1 NET1 SAP1: Fastq files for technical replicates prepared from the same library were pooled before alignment to improve the sensitivity of the analysis.
ChipSeq except total_h3: Basecalls performed using CASAVA version 1.4 & 1.5
ChipSeq except total_h3: Alignments performed using Eland for CASAVA version 1.4 & 1.5
ChipSeq total_h3: Basecalls performed using CASAVA version 1.8
ChipSeq total_h3: Alignments were performed using bwa version 0.5.9-r16
RNASeq: Alignments were performed using bwa version 0.5.9-r16
Genome_build: MM9
Supplementary_files_format_and_content: ChipSeq: Wig files are those generated by MACS at 1bp resolution and a set normalised to 15 million mapped reads is additionally provided.
Supplementary_files_format_and_content: ChipSeq total_h3: wig files generated by MACS at 1bp reolution
Supplementary_files_format_and_content: RNASeq: wig files generated by MACS at 1bp resolution
|
| |
|
| Submission date |
Apr 09, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Francesco Gualdrini |
| E-mail(s) |
francesco.gualdrini@crick.org.uk
|
| Organization name |
Francis Crick Institute
|
| Lab |
Treisman
|
| Street address |
44 Lincoln's Inn Fields
|
| City |
London |
| ZIP/Postal code |
WC2A 3LY |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL11002 |
| Series (1) |
| GSE45888 |
Definition of the fibroblast SRF-mediated immediate-early transcriptional response |
|
| Relations |
| SRA |
SRX262842 |
| BioSample |
SAMN02010512 |
