GSM5851339: Joint_HAO_K_3; Mus musculus; RNA-Seq
Ankle joints or ear pinnae were teased apart using forceps; excised LNs were passed through Nitex mesh (Cadisch Precision Meshes) and both tissues digested for 25 minutes at 37 °C with 2.64 mg/mL collagenase D (Roche). Joint tissue was then homogenized using the Miltenyi Tissue Dissociator and passed through a 70 μm cell strainer to obtain a single cell suspension. Joint and LN single cell suspensions were then washed in FACS buffer (PBS/ 2 % FCS/ 2mM EDTA) and stained for flow cytometry analysis. Cells stained for viability in PBS using Fixable Viability Dye (eFluor506; eBioscience) for 30 minutes at 4 °C. Cells were then washed in FACS buffer and subsequently incubated with FcR block for 10-15 minutes at 4 °C and then appropriate fluorochrome-conjugated antibodies (Supplemental Table 7) added and the cells stained for 30 minutes at 4 °C. Viable, single CD45+ Kaede Red+ cells were sorted using an Aria III (BD Biosciences). RNA libraries were prepared for sequencing using standard Illumina protocols Pair ended with a 100bp read length (Source Bioscience, Nottingham, UK).