GSM7743849: synovium from rat (rat ID #16) with medial meniscus transection surgery 4 weeks post-surgery; Rattus norvegicus; RNA-Seq
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).