GSM6569614: D28 post-depletion, WT tendon 1; Mus musculus; RNA-Seq
FDL tendons (1 tendon per biological sample; N=3 biological samples per genotype) were harvested at D28 post-surgery and flash frozen in liquid nitrogen. Total RNA was isolated using the Powermasher II (DIAGNOCINE) to homogenize the tissue. RNA was isolated from the extract using Trizol (Life Technologies, Carlsbad, CA) and the RNeasy Plus Micro Kit (Qiagen, Valencia, CA) per manufacturer's recommendations. The total RNA concentration was determined with the NanoDrop 1000 spectrophotometer (NanoDrop, Wilmington, DE) and RNA quality assessed with the Agilent Bioanalyzer (Agilent, Santa Clara, CA). The RNA integrity number (RIN) for all harvested samples was 7.73 ± 0.22 (mean ± standard deviation). The TruSeq Stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA) was used for next-generation sequencing library construction per manufacturer's protocols. Briefly, mRNA was purified from 200 ng total RNA with oligo-dT magnetic beads and fragmented. First-strand cDNA synthesis was performed with random hexamer priming followed by second-strand cDNA synthesis using dUTP incorporation for strand marking. End repair and 3` adenylation was then performed on the double-stranded cDNA. Illumina adaptors were ligated to both ends of the cDNA and amplified with PCR primers specific to the adaptor sequences to generate cDNA amplicons of approximately 200–500 bp in size. The amplified libraries were hybridized to the Illumina flow cell and single end reads were generated for each sample using Illumina NovaSeq6000. RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.