|
| Status |
Public on Nov 08, 2016 |
| Title |
RD_fibersMMP13_4h_1_CCTACC |
| Sample type |
SRA |
| |
|
| Source name |
RD_fibersMMP13_4h
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell line: Rat-1
cell type: fibroblast
treatment: MMP13
|
| Treatment protocol |
Rat-1 fibroblasts were seeded (25000 cells per well) in duplicates and incubated for 4 h at 37°C and 5% CO2. The cells adhering to ECM were extracted and used for mRNA profiling.
|
| Growth protocol |
Rat -1 fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 2 mM L-glutamine, 1% Penicillin/Streptomycin (Invitrogen) and 10% fetal bovine serum (FBS).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells adhering to native or degraded ECMs were directly lysed in the presence of QIAzol and total RNA was extracted with the miRNeasy Mini Kit (Qiagen). The RNA integrity number (RIN) was determined using the TapeStation System (Agilent Technologies). Quantity of RNA was determined by Qubit Fluorometric Quantitation kit (Life Technologies).
3' end mRNA libraries were prepared for sequencing using a method developed at Dr. Ido Amit's lab, Weizmann Institute of Science
3' RNA-seq for digital gene expression quantitation
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
single end protocol (R2 contain UMI of size 8 moved to fastq header): Fastq files contain data for alignment only on read 1. Sequencing used a short read on read 2, this read contain: 1) Sample barcodes 2) random sequence which we call UMI. Those 15 nucleotides were extracted from read 2 and concatenated to the fastq header of read 1.
|
| Data processing |
Illumina bcl2fastq software used for basecalling.
alignment: TopHat with default parameters
filter PCR amplification bias using alignment break site and UMI barcode (size 8) from R2 file.
The raw expression levels of the genes were calculated using counts obtained using the ESAT program (http://garberlab.umassmed.edu/software/esat/)
Normalization was done using DESeq
Genome_build: rn5
Supplementary_files_format_and_content: tab-delimited text file includes mRNA molecule count values for each Sample
|
| |
|
| Submission date |
Mar 31, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Ido Amit |
| E-mail(s) |
ido.amit@weizmann.ac.il
|
| Phone |
972-8-9343338
|
| Organization name |
Weizmann Institute of Science
|
| Department |
Immunology
|
| Street address |
234 Herzl st.
|
| City |
Rehovot |
| ZIP/Postal code |
760001 |
| Country |
Israel |
| |
|
| Platform ID |
GPL20084 |
| Series (1) |
| GSE79749 |
Distinct Combinatorial Events Generated by ECM Degradation Dictate Cell Behavior |
|
| Relations |
| BioSample |
SAMN04592499 |
| SRA |
SRX1671232 |
