|
| Status |
Public on Dec 27, 2018 |
| Title |
F_20W_2 |
| Sample type |
SRA |
| |
|
| Source name |
skin
|
| Organism |
Mus musculus |
| Characteristics |
tissue: fibrotic tissue
strain: C3H/HeJ mice
time point: 20 weeks
|
| Treatment protocol |
NA
|
| Growth protocol |
NA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Skin samples were macrodissected and snap frozen in liquid nitrogen. Samples were pulvarized in liquid nitrogen and RNA was isolated using Qiagen Rneasy.
Library prep and seq: All of the RNA samples were quantified using the qubit RNA kit (Life Technologies) on the Qubit 2.0 fluorometer. The RNA integrity was assessed using the Agilent RNA 6000 nano chip on the Agilent Bioanalyzer 2100. Each sample had a RIN score greater than 8.0 and 250ng of each sample was used for library prep. Libraries were prepared using the TruSeq Stranded mRNA library prep kit (Illumina). Briefly, 250 ng of RNA from each sample was purified using poly-T coated magnetic beads before being fragmented for first strand cDNA synthesis. Samples are reverse transcribed in the first strand cDNA synthesis step using random primers, then amplified using DNA Polymerase I in the second strand cDNA synthesis step. Samples were cleaned using AMPure beads before being single “A” base extended in preparation for adaptor ligation to generate indexed, cDNA libraries. Final cDNA libraries were size validated using the Agilent Bioanalyzer 2100 and quantified by qPCR (Kapa Biosystems). All libraries were normalized to 10nM, pooled 5 samples per pool and diluted down to 2nM. 2nM pools were denatured using 0.2N NaOH and diluted to a final concentration of 10 pM. 10pM pools were loaded onto the Illumina cBot for cluster generation. The clustered flow cell was sequenced paired-end 101 cycles using v3 reagents on the Illumina HighSeq 2000 to achieve a minimum of ~30 million reads per sample.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Overall read quality was checked using FASTQC v.0.11.2. Overall these plots showed that the base data off sequencer is of acceptable quality for analysis. For RNA QC the tool RNA-SeQC (v1.1.7) was used. Overall the RNA sequencing quality is acceptable for further analysis.
The raw sequence data, in the form of FASTQ files, was aligned to the human genome (mm10, iGenome GTF definition file) using the BOWTIE/TOPHAT pipeline (BOWTIE v2.2.3, TOPHAT 2.0.13). Accessory programs for the alignment stage include SAMTOOLS (v1.0) and cutadapt (1.7.1). Transcript assembly, abundance estimation, and tests for differential regulation were done using CUFFLINKS (v2.2.1).
Results of differential expression testing were loaded into the CummeRBund software in R (v3.1.1) for final output of results and graphing.
Genome_build: mm10
Supplementary_files_format_and_content: FPKM
|
| |
|
| Submission date |
Apr 25, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Benjamin Haibe-Kains |
| E-mail(s) |
benjamin.haibe.kains@utoronto.ca
|
| Phone |
+14165818626
|
| Organization name |
Princess Margaret Cancer Centre
|
| Department |
Princess Margaret Research
|
| Lab |
Bioinformatics and Computational Genomics
|
| Street address |
610 University Avenue
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 2M9 |
| Country |
Canada |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE98157 |
Fatty Acid Oxidation and Glycolysis Regulate Skin ECM Homeostasis by Shifting Fibroblasts between Catabolism and Anabolism (RNA-Seq) |
| GSE98160 |
Fatty Acid Oxidation and Glycolysis Regulate Skin ECM Homeostasis by Shifting Fibroblasts between Catabolism and Anabolism |
|
| Relations |
| BioSample |
SAMN06829623 |
| SRA |
SRX2763559 |
