GSM8746401: PCV2; Mus musculus; RNA-Seq
On the 28th day post infection, one mouse was randomly selected from the control group and the PCV2 group, and their spleens were prepared for preparation of single-cell suspension. Place a sterile RNase-free culture dish containing an appropriate amount of calcium-free and magnesium-free PBS on ice, the spleen tissue was transferred into the culture dish and cut it into 0.5 mm2 pieces, the tissues were washed with PBS, and remove as many non-purpose tissues as possible such as blood stains and fatty layers. Tissues were dissociated into single cells in dissociation solution (0.35% collagenase IV5, 2 mg/ml papain, 120 Units/ml DNase I) in 37 ℃ water bath with shaking for 20 min at 100 rpm. Digestion was terminated with 1× PBS containing 10% fetal bovine serum（FBS, V/V）, then pipetting 5-10 times. The resulting cell suspension was filtered by passing through 70-30 um stacked cell strainer and centrifuged at 300 g for 5 min at 4 °C. The cell pellet was resuspended in 100 ul PBS (0.04% BSA) and added with 1 ml 1× red blood cell lysis buffer（MACS 130-094-183, 10×）and incubated at room temperature or on ice for 2-10 min to lyse remaining red blood cells. After incubation, the suspension was centrifuged at 300 g for 5 min at room temperature. The suspension was resuspended in 100 μl Dead Cell Removal MicroBeads (MACS 130-090-101) and remove dead cells using Miltenyi ® Dead Cell Removal Kit (MACS 130-090-101). Then the suspension was resuspended in PBS (0.04% BSA) and centrifuged at 300 g for 3 min at 4 °C（repeat twice）. The cell pellet was resuspended in 50 μl of PBS (0.04% BSA). The overall cell viability was confirmed by trypan blue exclusion, which needed to be above 85%, single cell suspensions were counted using Countess II Automated Cell Counter and concentration adjusted to 700-1200 cells/μl. Single-cell suspensions were loaded to 10x Chromium to capture 5000 single cell according to the manufacturer's instructions of 10X Genomics Chromium Single-Cell 3' kit (V3). The following cDNA amplification and library construction steps were performed according to the standard protocol. Libraries were sequenced on an Illumina NovaSeq 6000 sequencing system (paired-end multiplexing run, 150 bp) by LC-Bio Technology co.ltd., (Hangzhou, China) at a minimum depth of 20,000 reads per cell.