|
| Status |
Public on Oct 22, 2016 |
| Title |
ns_pi3 |
| Sample type |
SRA |
| |
|
| Source name |
Unstretched monolayers pretreated with 1microM GSK2606414 PERK kinase inhibitor for 18 hours
|
| Organism |
Rattus norvegicus |
| Characteristics |
cell type: alveolar type-I-like epithelial (AEC-I) cells
source: primary isolated cells
strain: Sprague-Dawley
|
| Treatment protocol |
Prepared monolayers were randominzed into 4 conditions: 1. Unstretched membranes treated with 0.01% DMSO for 24 hours (NS). These monolayers served as controls. 2. Unstretched monolayers pretreated with 1microM GSK2606414 (PI) compound for 24 hours (NSPI). 3. Monolayers stretched with 25% surface area change for 6 hours after 18 hours of 0.01% DMSO treatment (S). 4.Monolayers stretched with 25% surface area change for 6 hours after 18 hours of 1microM PI treatment (SPI).
|
| Growth protocol |
Primary type II pneumocytes were grown fibronectin-coated silastic membranes. After 4 days of culturing the cells transform to AEC-I cells and form monolayers.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted from 20 rat AEC-I samples and DNase treated before library preparation for next generation sequencing. We pooled samples from 4 technical replicates of monolayers prepared from the same animal to provide sufficient genomic material for RNA extraction.
500ng of total RNA was used to generate libraries with Lexogen's QuantSeq 3'mRNA-Seq library prep kit for Illumina, FWD (Cat #015.24). The QuantSeq kit generates one fragment per transcript and sequences obtained are close to the 3'end of polyadenylated RNA. The quality of purified libraries was checked by Agilent Bioanalyzer using High Sensitivity DNA kit and quantified by KAPA library Quantification kit_Illumina (Cat # KK4835). Finally, equimolar concentrations of all libraries were pooled and subsequently next generation sequencing was performed on the NextSeq500 instrument (75 bp single read) using NextSeq(tm) 500 Mid Output Kit (cat#FC-404-2001).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
library strategy: Quant-Seq
|
| Data processing |
fastq files from 20 sequenced samples. N=5 biological replicates were used for 4 experimental conditions
STAR was used to align reads to the rn6 reference genome
HTSeq was used to obtain raw read counts per gene
DESeq2 was used to perform differential expression analysis
Genome_build: rn6
Supplementary_files_format_and_content: tab-delimited text files include raw read count values per gene for each sample that passed QC (Rat_Stretch_HTSeq_count.txt) and gene-based differential expression results for pairs of samples that passed QC under mechanical stretch and PERK inhibitor (PI) treatment (DESeq2_output_from_HTSeq_counts_controlling_for_rat.txt)
|
| |
|
| Submission date |
Oct 21, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Tamas Dolinay |
| E-mail(s) |
[email protected]
|
| Phone |
4125278764
|
| Organization name |
University of Pennsylvania
|
| Department |
Bioengineering
|
| Lab |
Susan Margulies
|
| Street address |
220 S. 33rd St., Towne building, Room 390
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL20084 |
| Series (1) |
| GSE89024 |
Quant Seq analysis of primary stretched rat alveolar type I-like epithelial (AEC-I) cells |
|
| Relations |
| BioSample |
SAMN05933186 |
| SRA |
SRX2256005 |
