|
| Status |
Public on Aug 30, 2022 |
| Title |
704CR |
| Sample type |
SRA |
| |
|
| Source name |
Knee OA synovium
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: primary FLS
treatment: 5 nM Cel-miR-39-3p (control) mimic for 48 h
|
| Treatment protocol |
Confluent OA FLS cultures are serum-starved for 3 h in DMEM/1% PS/0.5% FBS. Cells are then incubated with 3 µg/mL Lipofectamine RNAi MAX transfection reagent with 5 nM hsa-miR-27b-3p mimic (Qiagen #339173 YM00470553) or Control Mimic (Cel-miR-39-3p; Qiagen, #YM00479902) for 48h.
|
| Growth protocol |
FLS are dissociated from OA synovium of 3 separate patients undergoing total knee replacement surgery (Kellgren-Lawrence Grade III/IV) by sequential enzymatic digestion. Sub-confluent primary synoviocytes are passaged three times (P3) and grown in 10-cm petri dishes to confluency in DMEM/1% PenStrep/10% FBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA is extracted from control or mimic-treated OA FLS (n=3 per group) by standard Trizol method and quantified using the Qubit RNA BR assay (Denovix). Quality of RNA is assessed using the Agilent RNA Nano chips on a Bioanalyzer instrument. For all the samples, RNA integrity number (RIN) was >9.7.
Total RNA libraries are prepared from 200 ηg extracted RNA using the TruSeq Stranded Total RNA Library Preparation kit with RiboZero (Illumina Cat #20020596) according to manufacturer’s protocol. Individual libraries are quantified using a fluorometric high-sensitivity dsDNA assay (Denovix). Library quality is assessed on a high sensitivity DNA chip on the Agilent 2100 Bioanalyzer system (Agilent).
Libraries are prepared for sequencing (volumetrically pooled, diluted and denatured): 75 paired end read-cycles on an Illumina NextSeq 550 sequencer.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
processed data file: GTavallaee_TotalRNAseq_genecounts.tsv
|
| Data processing |
Demultiplexing of .bcl files and conversion into Fastq files is performed, followed by quality assessment for each sample using the bcl2fastq conversion tool (v2.19.1.403).
Cutadapt (v1.18) is used to maintain minimum read length of 25 bp post trimming of adapters as well as trimming of N’s.
HISAT2 software (v2.1.0 with --rna-strandness RF –dta parameters) is used to perform splice-aware alignment of reads using a Hierarchical Graph FM index method against human reference genome (vGRCh38).
StringTie software (v1.3.5 with parameters --rffor stranded library fr- firststrand) is used to assemble the aligned reads into transcripts.
StringTie (parameters -e -B -G referencetranscripts.gtf) is used to obtain the abundance of the transcripts.
‘prepDE.py’ by StringTie software is used to extract the read count information.
Genome_build: GRCh38
Supplementary_files_format_and_content: GTavallaee_TotalRNAseq_genecounts.tsv: Tab-delimited text file includes raw count values for each Sample.
|
| |
|
| Submission date |
Jun 16, 2020 |
| Last update date |
Sep 08, 2022 |
| Contact name |
Mohit Kapoor |
| E-mail(s) |
mohit.kapoor@uhnresearch.ca
|
| Organization name |
Schroeder Arthritis Institute
|
| Street address |
60 Leonard Ave
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5T 0S8 |
| Country |
Canada |
| |
|
| Platform ID |
GPL21697 |
| Series (1) |
| GSE152638 |
Effect of microRNA-27b-3p mimic treatment on human osteoarthritis fibroblast-like synoviocyte gene expression profiles |
|
| Relations |
| BioSample |
SAMN15287037 |
| SRA |
SRX8562168 |
