RNA-seq of synovial compartment separated by Laser Capture Microdissection (LCM).
Synovium was identified by characteristic histologic features. Frozen samples were cryosectioned using a Thermo Scientific CryoStar NX70 at -20°C. Three to four slices of 16 µm thickness were transferred to uncharged glass slides. Staining was performed after removing slides from -80°C and immediately applying RNAprotect Tissue Reagent (Qiagen, #76104).  Tissues were fixed in 75% ethanol prepared with UltraPure water (Invitrogen, #10977015). Slides were dunked in UltraPure water with ProtectRNA RNase Inhibitor (Sigma, # R7397). Slides were stained with Arcturus™ Histogene™ LCM Frozen Section Staining Kit (Applied Biosystems, #KIT0401). Slides were immediately rinsed in water with RNase inhibitor. Tissues were dehydrated with a series of ethanol and xylene immersions. A single layer of desiccant was added to the bottom of the staining jars containing 100% ethanol and slides were allowed to air dry at room temperature. Slides were placed on an ArcturusXT Laser Capture Microdissection system. A CapSure MacroCap (Applied Biosystems, #LCM0212) was placed onto each tissue section. Substructures were collected in sequential order (lining, vessels, sublining) by firing the infrared laser through the top of the cap, with one collection cap, substructure, and slide. Caps with collected tissue were incubated at 42°C in 30 µl PicoPure RNA Isolation Kit (Applied Biosystems, #KIT0204) extraction buffer. Lysates were stored at -80°C prior to RNA isolation using the PicoPure RNA Isolation Kit. Genomic DNA was eliminated by on-column DNase treatment using the RNase-Free DNase Set (Qiagen, #79254). RNA concentration was evaluated with an Agilent Bioanalyzer 2100, using the RNA High Sensitivity Assay (Agilent, #5067-5579, #5067-5581, #5067-5580). Sequencing libraries were constructed using the SMARTer Stranded Total RNA-Seq Kit v3 (Takara, #634487), including fragmentation and ribosomal RNA depletion steps. cDNA library quality and concentration were evaluated with an Agilent Bioanalyzer 2100, using the D5000 DNA assay (Agilent, #5067-5588, #5067-5589, #5067-5590).