title: WSX1-D10-E-RK
organism: Mus musculus
description: Protocols: The design and synthesis of CpG-Stat3-siRNA oligonucleotide is described elsewhere (PMID:19749770). Forward and reverse oligonucleotides (50 nmol) were dissolved in 250μl of DNase/RNase-free water at 37°C for 5-7 minutes before complementary hybridization by heating to 80°C for 1 minute and returning to 37°C for 1 hour.  Mice were challenged (s.c.) with methylated BSA (mBSA; 1 mg/ml emulsified in Complete Freund's Adjuvant) and 160 ng Bordetella pertussis toxin (i.p.). Following an antigen boost with mBSA and CFA (s.c.), inflammatory arthritis was established by intra-articular (i.a.). Mice were administered (i.a.) with CpG-Stat3siRNA (0.125 nmol/μl) at the point of arthritis induction in combination with mBSA (total volume of 10μl/joint). To test the specificity of this treatment, mice with AIA were also administered at the point of arthritis induction with either CpG alone or a non-hybridized, single-stranded version of CpG-Stat3siRNA (confirmed by histological assessment alone).  Synovial tissues were harvested on days 0, 3, and 10 of AIA. Total RNA was isolated from tissue extracts using RNeasy Mini kits (QIAGEN). Libraries were generated from 2-4mg of mRNA following removal of cytoplasmic, mitochondrial, and ribosomal RNA (ribominus transcriptome isolation kit; Ambion). Libraries were prepared using the TrueSeq Stranded Total RNA library Prep kit (Illumina) or RNA-seq kit v2 (Life Technologies; 4475936) and sequenced on an Illumina HiSeq 4000 platform. RNA was extracted utilising QIAshredders (Qiagen) and RNeasy Mini Kits (Qiagen). On-column DNase digestion (Qiagen) was utilised to remove contaminating genomic DNA. Single-stranded cDNA was synthesized, processed and labelled sequentially using the Ambion® WT (Whole Transcript) Expression Kit, the Affymetrix® Genechip® Poly-A RNA Control Kit and the Terminal Labelling kit.