 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 20, 2018 |
| Title |
control_2_3'SAGE-seq |
| Sample type |
SRA |
| |
|
| Source name |
control_3'SAGE-seq
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6
genotype/variation: Col1a2-GFP Transgenic
genotype/variation: mock control
tissue: lung
cell type: lung fibroblast
cell type: lineage (CD45, CD31, EpCAM, CD146 and Ter119) negative, Col1a2-GFP and LNGFR positive fibroblast
|
| Treatment protocol |
C57BL/6 mice were anesthetized with isoflurane before a single dose of bleomycin sulfate (2 mg/kg dissolved in 50 μL of sterile saline solution; Toronto Research Chemical, Toronto, ON, Canada) was instilled by oropharyngeal aspiration. Primary fibroblasts were isolated from Col1a2-GFP murine lungs and miR-20a and control vector (marker : hLNGFR) were retrovirally transduced. 5x10^6 of each transduced cell were intratracheally-transferred to B6 mice at day 7 post-administration of 2 mg/kg of bleomycin.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Transferred lung fibroblasts were isolated from bleomycin-treated lungs of C57BL/6 mice at day 3 post-transfer. Lungs were cut into small pieces with a razor blade and digested enzymatically using 0.2% collagenase (Wako, Osaka, Japan), 0.96 mg/mL Dispase II (Roche, Basel, Switzerland), and 20 kU/mL DNase I (Sigma-Aldrich, St Louis, MO) for 60 min at 37°C. Erythrocytes were removed by 70% Percoll (GE Healthcare, Buckinghamshire, UK) density separation. The single-cell suspensions were then depleted of lineage+ (CD31+, CD45+, EpCAM+, CD146+ and Ter119+) cells by negative selection with an AutoMACS cell separator (Miltenyi Biotech, Bergisch Gladbach, Germany). Lineage− Col1a2GFP+ hLNGFR+ fibroblasts were further purified by cell sorting using a FACS Aria II (BD).
polyA RNAs were isolated and amplified from donor fibroblasts according to the protocol described in (Huang H, Nucleic Actd Res, 2014) with some modifications. 3'SAGE-seq libraries were constructed according to the protocol described in (Matsumura H, Methods Mol Biol, 2012) with some modifications. Sequencing was performed by using Ion Hi-Q Chef Kit, Ion PI v3 Chip Kit and Ion Proton Sequencer (Thermo Fisher Scientific) according to the manufacture’s instructions except the input library concentration (100 pM) and cycle number (200).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent Proton |
| |
|
| Data processing |
Adapter trimming and quality filtering of sequencing data were performed by using Trimommatic-v0.36 and PRINSEQ-0.20.4. Filtered reads were mapped to Refseq mm10 by using Bowtie2-2.2.5. Reads that were not mapped to NlaIII sites were removed, and quantified tag numbers of each gene as the expression level of each gene.
Total tag number was adjusted to 1 million tags, and between-sample normalization was performed by using R-3.1.1 and TCC package.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include normalized tag-count values for each sample
|
| |
|
| Submission date |
Jun 16, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Shigeyuki Shichino |
| E-mail(s) |
[email protected]
|
| Organization name |
Tokyo University of Science
|
| Department |
Department of Molecular regulation of Inflammatory and Immune Diseases
|
| Street address |
2641, Yamasaki, Noda-shi
|
| City |
Chiba |
| ZIP/Postal code |
278-0022 |
| Country |
Japan |
| |
|
| Platform ID |
GPL18635 |
| Series (2) |
| GSE100116 |
Transcriptomic analysis of miR-19a-19b-20a subcluster-overexpressed fibroblasts in bleomycin-induced lung fibrosis |
| GSE100147 |
Global miRNA expression profiling of lung fibroblasts identifies miR-19a-19b-20a subcluster as a suppressor of TGF-beta-associated fibroblast activation in murine pulmonary fibrosis |
|
| Relations |
| BioSample |
SAMN07249388 |
| SRA |
SRX2925255 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2671491_control_2.txt.gz |
124.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
