GSM6189229: Achilles tendon-derived cells, vehicle treatment, replicate 1 [E1]; Rattus norvegicus; RNA-Seq
Tendon-derived fibroblasts utilized in this study were from a previous investigation (Patel et al. 2019 Sci Reports). Briefly, Achilles tendons were rinsed with sterile PBS, minced, placed in DMEM containing 0.2% type I collagenase, and incubated in a 37°C shaking water bath for four hours. After digestion, the cell suspension was filtered through a 100μm mesh filter, pelleted by centrifugation, and resuspended in 5.5mM glucose DMEM containing 10% FBS, 1% sodium pyruvate (Sigma, St. Louis, MO), and 1% penicillin/streptomycin (Thermo Scientific, Waltham, MA). Samples were then plated in 100mm collagen-coated dishes. After reaching confluency, tendon fibroblasts were split and seeded (100,000 cells) in 100mm collagen-coated culture plates. Libraries from 500ng of input total RNA were constructed as directed by the Nugen Universal Plus mRNA-Seq + UDI kit (PN#9144-96), but the RNA fragmentation time was decreased from 8 minutes to 4 minutes. Final library products were subjected to a 0.7 Ampure:1 Sample ratio purification to reduce lower molecular weight amplicons. The resulting libraries were assessed with an Agilent DNA High Sensitivity Chip for yield and quality and sequenced by Novogene (Sacramento, CA). Ten libraries were pooled and evenly distributed across a single HiSeq lane to generate ~40,000,000 2X150bp reads on the HiSeq 4000 platform (Illumina, San Diego, CA).