GSM7184313: SPF_3; Sus scrofa; RNA-Seq
Adipose tissue were transported in In sterile culture dish with 10 ml 1× DPBS solution (Solarbio, cat. no. D1040) on ice  to remove the residual MACS Tissue Storage Solution (Miltenyi Biotec, cat. no. 130-100-008), then minced on ice.  We used dissociation enzyme 0.25% Trypsin (Hyclone, cat. no. SH30042.01) and 10 ug/mL Recombinant DNase I  (RNase-free) (TaKaRa, cat. no. 2270B) dissolved in DPBS with 5% Fetal Bovine Serum (FBS, Thermo Fisher  Scientific, cat. no. SV30087.02) to digest the tissues. XX tissues were dissociated at 37 ℃ with a shaking speed of  50 r.p.m for about 40 min.We repeatedly collected the dissociated cells at interval of 20 min to increase cell yield  and viability. Cell suspensions were filtered using a 40 μm nylon cell strainer and red blood cells were removed by  1× Red Blood Cell Lysis buffer (Thermo Fisher Scientific, cat. no. 00-4333-57) . Dissociated cells were washed  with 1× DPBS containing 2% FBS. Cells were stained with AO/PI, and viability was assessed using a Countstar  Fluorescence Cell Analyzer (Aber Instruments). Beads with unique molecular identifier (UMI) and cell barcodes were loaded close to saturation, so that each cell  was paired with a bead in a Gel Beads-in-emulsion(GEM). After exposure to cell lysis buffer, polyadenylated RNA  molecules hybridized to the beads. Beads were retrieved into a single tube for reverse transcription. On cDNA  synthesis, each cDNA molecule was tagged on the 5'end (that is, the 3'end of a messenger RNA transcript) with  UMI and cell label indicating its cell of origin. Briefly, 10× beads that were then subject to second-strand cDNA  synthesis, adaptor ligation, and universal amplification.Sequencing libraries were prepared using randomly  interrupted whole-transcriptome amplification products to enrich the 3' end of the transcripts linked with the cell  barcode and UMI. All the remaining procedures including the library construction were performed according to the  standard manufacturer's protocol (Chromium Single Cell 3ʹ v3.1). Sequencing libraries were quantified using a  High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 and the Qubit High Sensitivity DNA Assay (Thermo  Fisher Scientific). The libraries were sequenced on NovaSeq6000 (Illumina), and 150 bp paired-end reads were  generated. The sequencing and bioinformatics analysis were performed by Majorbio Co., Ltd (Shanghai, China).