GSM6046123: MMS synovium; Mus musculus; RNA-Seq
GSM6046123
Synovium from control and MMS D14 mice knees (20 knees respectively) were dissected, minced with scissors and transferred into RPMI-1640 (Nacalai Tesque) containing 0.2mg/ml Liberase TL (Sigma-Aldrich) and 0.1mg/ml DNaseⅠ (Roche) at 37℃ for 20 min with intermittent syringe homogenization. Cells were filtered through 100 µm and 40 µm cell strainers and 40 µm Flowmi cell strainer (Sigma-Aldrich). For each synovium sample, single cell RNA libraries were generated using the 10x Genomics Chromium platform. Single cell suspensions were loaded onto the Chromium Single Cell system v3.1 (Ghromium Next GEM Single Cell 3' Reagent Kits v3.1). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). Following the GEM generation, the Gel bead is dissolved, primers are released, and any co-partitioned cell is lysed. Primers containing the poly(dT) sequence, Ilumina TurSeq Read1, Unique Molecular Identifier (UMI) and the 10x Barcode are mixed with the cell lysate and a Master Mix containing reverse transcription reagents. After incubation, the pooled barcoded cDNA was then cleaned up with DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction, Ilumina TruSeq Read2, P5, P7, i7 and i5 sample index were added via End Repair, A-tailing, Adaptor Ligation, and PCR.