GSM6260078: Skin_healthy_mouse_6_CIA; Mus musculus; RNA-Seq
All disected organ samples were cryopreserved in 10% DMSO and 90% FBS until further analysis. For sequencing, the samples were quickly thawed in a 37 ºC water bath with continuous agitation, then transferred into 15 mL centrifuge tube with 1 mL pre-warmed thaw solution (90% Hibernate-A and 10% FBS) and incubated at room temperature for 1 minute. Then 2 mL, 5 mL and 5 mL thaw solution were added into the centrifuge tube, separated by a 1-minute incubation. Next, the samples were centrifuged at 350 × g for 5 minutes. Lastly, the samples were resuspended with 1 mL Hibernate-A, after which the supernatant was removed and incubated until next step.   Samples from spleen were squeezed to pass through 70 µm strainer to collect cells in a 50 mL centrifuge tube. After being centrifuged at 350g for 5 minutes, cells were resuspended with 5mL red blood cell lysis buffer for 5 minutes. Lysis reaction was quenched by adding medium (90% RPMI-1640 with 10% FBS). Cells were centrifuged at 350 × g for 5 minutes and washed thrice to remove the lysis buffer. Single cells suspensions were prepared by RPMI-1640 at a density of 1 × 105 cells/mL. Samples from joint, muscle, lung, and skin were quickly transferred into 75mm dishes with 1mL DMEM after thawing, and then minced into ~1mm pieces with scissors. Next, pieces of organ samples were transferred into 15 mL centrifuge tube with 5 mL of DMEM and treated with enzymes (joint: 1.5 mg/mL Collagenase Ⅳ / DnaseⅠ; lung: 1.8 mg/mL Collagenase Ⅳ / DnaseⅠ; muscle: 2 mg/mL Collagenase Ⅱ / DnaseⅠ; skin: 2.6 mg/mL Collagenase Ⅰ/ DnaseⅠ) for 120 minutes. After dissociation, another 5 mL DMEM with 10% FBS was added into the 15mL centrifuge tubes. Then dissociated cells were centrifuged at 350 × g for 5 minutes after passing through 70 µM strainer, after which the cells were washed thrice with PBS. Single cells were re-suspended into RPMI-1640 at a density of 1 × 105 cells/mL for cell loading. All scRNA-seq experiments were then performed using the Seq-Well technique (Gierahn et al., 2017). Briefly, single cell suspensions were counted and co-loaded with barcoded and functionalized oligo-dT beads (Chemgenes, USA; cat. no. MACOSKO-2011-10) on microwell arrays. For each sample, 20,000 live cells were loaded per array, and libraries from three samples were pooled together for sequencing, resulting in a coverage of 6,6 reads per base. The microwell arrays were then covered with previously plasma treated polycarbonate membranes, and the membranes were allowed to seal to the bead and cell co-loaded microwell arrays at 37°C for 30 minutes. Next, cell lysis and hybridization were performed, followed by bead removal, reverse transcription, and whole transcriptome amplification. Libraries were prepared for each sample using the Nextera XT DNA Library Preparation Kit (Illumina, USA; cat. no. FC-131-1096) according to manufacturer's instructions. Libraries were prepared from GRCm38 (June 2017, Ensembl) using STAR software.