 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 05, 2021 |
| Title |
Ad-shRNA Cklf1 rep3 |
| Sample type |
SRA |
| |
|
| Source name |
Rat vascular smooth muscle cells
|
| Organism |
Rattus norvegicus |
| Characteristics |
Sex: male
genotype/variation: knockdown of Cklf1
strain: Sprague-Dailey
|
| Growth protocol |
Vascular smooth muscle cells with passage number four were transfected with overexpression of CKLF1 adenovirus particles overnight and then cultivated in the complete media. Until the confluence reached 75%, the cells in each well was starved with the media supplemented with 0.2% FCS for next 24 - 32 h. An adenovirus carrying green fluorescence protein was used as the negative control. For cells carrying with shRNA Cklf1 or shRNA Scramble, an additional treatment with 25 μg/L TNFɑ for 24 h were performed.Total RNA were extracted and generated by deep sequencing, in triplicate, on an Illumina X Ten platform.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA of 12 samples were isolated from adenovirus treated primary vascular smooth cells using Trizol reagent (Invitrogen) according to the manufacturer's instruction.
The total RNA samples are first treated with DNase I to degrade any possible DNA contamination. Then the mRNA is enriched by using the oligo(dT) magnetic beads. Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then the first strand of cDNA is synthesized by using random hexamer-primed reverse transcription. Buffer, dNTPs, RNase H and DNA polymerase I are added to synthesize the second strand. The double strand cDNA is purified with magnetic beads. End reparation is then performed. After the previous step, adaptors are ligated to the end of these fragments. Next, ligation products were selected by size and purified on TAE-agarose gel. Finally, the fragments are enriched by PCR amplification, then purified by magnetic beads and dissolved in the appropriate amount of Epstein-Barr solution. During the QC step, Agilent 2100 Bioanaylzer are used to qualify and quantify of the sample library. The library products are ready for sequencing via Illumina X Ten platform.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
| |
|
| Data processing |
Trimmed, clean reads were mapped to the rat reference genome (Rnor_6.0) using the SOAP2 software and allowing five mismatches(Li et al., 2009).
Fragments per kilobase of transcript per million fragments mapped (FPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
A FDR of no more than 0.05 (FDR ≤ 0.05) and a difference ratio of the FPKM between controls and CKLF1 samples of no less than 1 (|log2 ratio| ≥ 1) were adopted as the significance thresholds to identify DEGs.
Genome_build: Rnor_6.0
Supplementary_files_format_and_content: txt files include FPKM values for each Sample.
|
| |
|
| Submission date |
Mar 05, 2018 |
| Last update date |
Mar 06, 2021 |
| Contact name |
Yanyu Dean |
| E-mail(s) |
yanyuduan@gmail.com
|
| Phone |
86-791-83813080
|
| Organization name |
Jiangxi Agricultural University
|
| Lab |
State Key Laboratory of Pig Genetic Improvement and Production Technology
|
| Street address |
Lushanzhong888, Changbei district
|
| City |
Nanchang |
| ZIP/Postal code |
330044 |
| Country |
China |
| |
|
| Platform ID |
GPL24688 |
| Series (1) |
| GSE111398 |
RNA-Seq analysis, transcriptome assembly and gene expression profile analysis for vascular smooth muscle cells with up- or downregulation of CKLF1 |
|
| Relations |
| BioSample |
SAMN08635009 |
| SRA |
SRX3763995 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3030101_shRNA_CKLF1-3.txt.gz |
171.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
