title: Illumina HiSeq 4000 sequencing; Single RNA-Seq of synovial tissue macrophages from Rheumatoid Arthritis patients with active disease and in disease remission.
design: Single RNA-Seq of synovial tissue macrophages from Rheumatoid Arthritis patients with active disease and in disease remission.
library_construction_protocol: fresh synovial tissues were diced and reduced to small pieces around 1-2 mm3 fragments with Sterile Disposable Scalpels either No. 22. Once processed, tissue pieces are transfer into a sterile universal container containing 10ml sterile RPMI with Penicillin/Streptomycin and L-Glutamine (RPMI medium) in 1/33 dilution of Liberase TM Research Grade (0.15mg/ml, 0.78 Wunsch units/ml) (TM Research Grade (Thermolysin, Medium) from Roche Diagnostics (000000005401127001, Sigma). Tissue was incubated at 370C, 5% CO2 in a moist atmosphere 30-45 minutes while rotating on a Miltenyi MACSmix tube rotator and shook vigorously by hand twice during incubation. After incubation, the digested mixture was filtered using an Easy Strain 100μM into 50ml falcon tube. Then, the original container was washed with the complete media (RPMI/10%FCS/L-glutamine/Penicillin/Streptomicin) and the wash was filtered through. The end (rubber) of a 1ml syringe was used to massage gently the surface of the filter. Further complete media was poured through the filter into the falcon tube up till 40ml and everything was spun down at 1800 RPM for 10 min at 4oC. Then, the supernatant was carefully removed. One ml of complete media was added to the cell pellet to gently resuspend cells using 1ml tip with the wide opening to avoid cell shredding and the cells were moved to a sterile Eppendorf.  Twenty ul aliquot was used to count the cells. Cells were spun down at 1500 RPM for 5 min at 4oC. The supernatant was removed, and cells were used for STM phenotyping and/or STM population sorting as described below. Alternatively, to digested tissues from 25 patients/healthy donors 1ml of Bambanker (302-14681; Wako) was added on ice using the tip with a wide opening and cells were immediately frozen at -80 oC and subsequently stored in liquid nitrogen to the day of scRNAseq experiment. Synovial tissue macrophages were sorted from digested synovial tissue biopsies based on the positive expression of CD11b and CD64 and negative expression of a range of other lineage specific cell markers (CD3, CD19, CD20, CD56, CD49, CD117 and CD15). Around 2000-10000 synovial tissue macrophages per sample were sorted into qPCR 0.2ml tubes coated with FSC and containing 10ul of PBS/0.02%BSA according to 10xGenomics protocol. Perform through 10X genomics Perform through 10X genomics
attributes.tag: Experimental Factor: disease
attributes.value: undifferentiated arthritis
attributes.tag: Experimental Factor: response to treatment
attributes.value: undifferentiated arthritis