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| Status |
Public on Aug 29, 2023 |
| Title |
cartilage from rat (rat ID #11) with sham surgery 4 weeks post-surgery |
| Sample type |
SRA |
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| Source name |
cartilage
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| Organism |
Rattus norvegicus |
| Characteristics |
tissue: cartilage
timepoint: 4 weeks
disease state: Sham Surgery
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| Treatment protocol |
All animal procedures were approved by the Scripps Research Institutional Animal Care and Use Committee. Male Lewis rats (n=42 total) were assigned to the following groups: 24 rats with MMT surgery on the right and sham surgery (SS) on the left knee for knee tissue collection and RNA sequencing at 2, 4 and 6 weeks (n=8 animals per time point); 18 rats with MMT surgery on the right and sham surgery on the left knee for knee histology at 2, 4 and 6 weeks (n=6 animals per time point). Animals were anesthetized with ketamine/xylazine cocktail (50 mg/kg Ketaved – Henry Schein, and 5 mg/kg Xylazine – AniSed respectively) by intraperitoneal injection, administered appropriate analgesics (Flunixin, 5 mg/kg subcutaneous injection), and the surgical site shaved and disinfected. A small incision was made on the medial right hind knee to expose the collateral ligament. The collateral ligament was transected, and the joint capsule opened sufficiently to expose the medial meniscus. The meniscus was transected completely, then the skin incision was closed, and pressure applied to the wound to prevent hematoma development. Animals were allowed to recover on a heated blanket and were returned to their home cage. Sham procedures were carried out in a similar fashion except the medial meniscus was not cut.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Rats were euthanized 2 (n=8), 4 (n=8), or 6 (n=8) weeks after MMT. Cartilage from both the medial and lateral sides of tibial plateaus and femoral condyles, medial and lateral menisci, and synovium of both knees were collected. All visible synovium tissue was collected from the medial and lateral sides of the joint capsule and femoral condyles, distal to the meniscus and directly adjacent to the tibial plateau at all time points. Due to the very thin layer of synovium, it was not possible to collect exclusively the lining layer without some sub synovial adipose tissue. Tissues were homogenized with 350 uL TRIZOL using a homogenizer, for three 30 s intervals interrupted b y3 min breaks. The homogenized tissue was added to 70 uL ethanol. This mixture was vortexed, and the 100 uL supernatant was collected, centrifuged, and added to the mRNeasy Mini kit column (Qiagen) following manufacturer's instructions. Total RNA was eluted in 12 uL RNase-free water. The RNA integrity was determined using and Agilent Bioanalyzer and only samples with values >5 were used for RNA-seq.
The high throughput RNA-seq assay used was part of an early access kit from iGenomX Inc. Briefly, total RNA (50 ng) was placed in a solution containing 1.4X First Strand Buffer and barcode oligo dT primer and incubated at 80C for 10 min in 7 uL total volume, subsequently added was 4.5 mM DTT, barcoded template-switching oligonucleotide, 1.8 U/uL RNaseOUT (ThermoFisher), dNTP mix and Reverse Transcriptase in 11 uL final volume. Reactions were incubated 50 min at 42C, followed by 5 min at 85C. This was followed by addition of 2 uL reaction clean-up mix and incubated at 37C for 30 min and 80C for 20 min. Samples were pooled together, and cDNA products were purified by 1.2X SPRI bead clean-up procedure with SPRI bead mixture. cDNA products were taken directly into a PCR reaction containing 1X KAPA HiFi reaction mixture and 0.5 uM barcoded PCR primers of Illumina p5 and p7 sequences in 100 uL final volume. PCR products were first cleaned using 1X SPRI bead clean-up mix, and then separated on 2% agarose gels. Products approximately 350 bp-800 bp were excised and isolated using agarose gel dissolving buffer and clean-up and concentrator column (Zymo Research). Purified PCR products were quantified using Qubit dsDNA HS assay kit, analyzed on Agilent Tapestation, and sequenced on 100 cycle P2 flow cells with an Illumina NextSeq2000 sequencing instrument using paired-end sequencing (read1: 26 bases, read2: 94 bases, i5 and i8 indexes used 8 base reads).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
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| Description |
03exp3X3RAT-sample061
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| Data processing |
The high throughput RNA-seq data was initially processed with in-house custom scripts using BBTools (https://sourceforge.net/ projects/bbmap/) to obtain demultiplexed reads for each sample based on the inline barcodes. The 5’-end 10-base barcodes and 3’-end 12-base UMI information from read1 fastq were first incorporated into read2 fastq as part of sequence ID using UMI-tools. The read2 sequences (from the pre-deduplicated dataset) were analyzed using nf-core/RNA-seq pipeline v1.4.2 (implemented on Nextflow v20.07.1), which is an open-source and available at https://github.com/nf-core/rnaseq as part of the nf-core project. Reads2s were trimmed for adapters using trimGalore! V0.6.4 (https://www.bioinformatics.babraham.ac.uk/projects/trim_ galore/) and aligned to the rat genome (Rattus norvegicus) ENSEMBL build Rnor 6.0 using STAR v2.6.1d. Gene-level assignment was then performed using featurecounts v1.6.4. The gene expression matrix with raw gene counts was then used for differential gene expression analysis using the Bioconductor DESeq2 R package v1.20.0. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate (FDR). Genes with an adjusted P-value (padj) < 0.05 found by DESeq2 were assigned as significantly differentially expressed genes (DEGs). The uniquely mapped alignments resulting from alignments to the reference genome were deduplicated removing the PCR duplicates. The transcript UMI for each gene was counted for each sample using UMI-tools.
Assembly: Rattus norvegicus (Rnor 6.0)
Supplementary files format and content: gene counts matrix, FPKM matrix, TPM matrix
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| Submission date |
Aug 29, 2023 |
| Last update date |
Aug 29, 2023 |
| Contact name |
Padma Natarajan |
| E-mail(s) |
ccbb@scripps.edu
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| Organization name |
The Scripps Research Institute
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| Department |
CCBB
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| Street address |
10550 N.Torrey Pines Road
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL32190 |
| Series (1) |
| GSE241794 |
Transcriptomic analyses of joint tissues during osteoarthritis development in a rat model reveal dysregulated mechanotransduction and extracellular matrix pathways |
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| Relations |
| BioSample |
SAMN37178126 |
| SRA |
SRX21497405 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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