GSM5287445: single-cell RNA seq of early weaning piglet_EWP3; Sus scrofa; RNA-Seq
The tissue was dissociated into 5 ml of digestion solution (0.35% collagenase IV5, 2 mg/ml papain, 120 unit/ml DNase I) in 37 °C water bath with shaking for 20 min at 100 rpm. Digestion was terminated with the PBS containing fetal bovine serum (FBS, 50% v/v), then pipetting 6 times with Pasteur pipette. The resulting cell suspension was filtered by passing through 70 μm stacked cell strainer and centrifuged at 300g for 5 min at 4 °C. The cell pellet was resuspended in 100ul PBS (0.04% BSA) and with 1 ml 1 × red blood cell lysis buffer (MACS, 10×) and incubated at 37 °C for 5 min to lyse remaining blood cells. And then, the suspension was resuspended in 50 μl of 1× PBS (0.04% BSA) after removing dead cells by Miltenyi® Dead Cell Removal Kit (MACS, 10× Genomics). Single-cell suspension was loaded to 10x Chromium to capture appropriate amount of single cell according to the manufacturer instructions of 10x Genomics Chromium Single-Cell 3' kit (V3). The following amplification of cDNA and library construction was performed per the standard manufacture's protocol. Libraries were subjected to an Illumina NovaSeq 6000 sequencing system (paired-end multiplexing run,150 bp)