alias: E-MTAB-4294:TRI3
title: Illumina HiSeq 2000 paired end sequencing; RNA-seq of synovial membranes from a preclinical model of post-traumatic knee osteoarthritis to evaluate the effects of corticosteroid treatment
design: RNA-seq of synovial membranes from a preclinical model of post-traumatic knee osteoarthritis to evaluate the effects of corticosteroid treatment
library_construction_protocol: 18 adolescent female Yorkshire pigs (E.M. Parsons & Sons Inc., Hadley, MA), aged 83-96 days and weighing 31-37 kg, were randomly assigned to one of three groups:  ACL transection (ACLT), ACL transection followed by injection of 20 mg triamcinolone acetonide injection (STEROID), and no surgery (INTACT control; n=6 for all groups).  In the ACLT and STEROID groups, the ACL was transected at the junction of the proximal and middle thirds as previously described. No post-operative immobilization was used. Pigs were housed in single cages with ad libitum access to water and were fed three times daily. No adverse event was observed throughout the experiment. After 14 days, the animals were euthanized and synovial fluid was drawn from the joints. Adjacent biopsies of the lateral synovial membrane (distant from the site of the original surgery) were collected, placed into sterile tubes (for RNA extraction) or embedded in optimal cutting temperature medium (OCT; Sakura Finetek, Torrance, CA) (for histology), frozen in liquid nitrogen and stored at -80C until processed for further analysis.  Total RNA was extracted from the synovial membranes through homogenization, phenol-chloroform separation and on-column purification using the PureLinkTM RNA Mini Kit (Life technologies, Carlsbad, CA). RNA samples were treated with DNase I (PureLinkTM DNase Set, Life technologies, Carlsbad, CA) and assessed with the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The samples were then enriched for polyA+ messenger RNA, reverse transcribed with random hexamers, ligated with indexed adapters and amplified with 15 cycles of PCR using the TruSeq RNA Sample Preparation Kit v2 (Illumina, San Diego, CA).
identifiers.namespace: Department of Orthopaedic Surgery, Boston Childrens Hospital, Harvard Medical School, Boston, MA, USA
identifiers.id: E-MTAB-4294:TRI3