title: Illumina HiSeq 2500 paired end sequencing; RNA-Seq of anterior cruciate ligament and knee joint synovium in dogs that are affected with cruciate rupture compared to matched controls.
design: RNA-Seq of anterior cruciate ligament and knee joint synovium in dogs that are affected with cruciate rupture compared to matched controls.
library_construction_protocol: Tissues from cases were collected during knee stabilization surgery. Tissues from unaffected control dogs were collected from dogs undergoing pelvic limb amputation or euthanasia for reasons unrelated to this study. Tissues were placed in RNALater and frozen until RNA extraction.  RNA was extracted using the Qiagen RNAeasy fibrous tissue kit with water as buffer. Samples were treated with DNase. TruSeq stranded mRNA libraries were prepared with polyA enrichment. Fragmented, primed, and finished using FPF mix. In line controls were included for all reactions. Samples were purified using Agencourt AMPure XP beads bead purification. Each sample underwent 10 PCR cycles. Qubit Quantification was performed with 1:100 dilution of library in singlet with DNA HS Rgt before libraries were submitted for final QC and sequencing.