title: GSM8145272: rat_fascia2; Rattus norvegicus; RNA-Seq
library_construction_protocol: The process of tissue dissociation and cell purification for single-cell RNA sequencing and SF primary cell extraction was meticulously executed. The SF and skin tissues were placed in a sterile culture dish containing 10 ml of 1x Dulbecco's Phosphate-Buffered Saline (DPBS; Thermo Fisher, Cat. no. 14190144) on ice to eliminate the residual blood and adipose, followed by mincing on ice. We employed a specially formulated dissociation solution, composed of the collagenase type 4 (sigma-Aldrich, Cat. no. C5138), elastase (sigma-aldrich, Cat. no. e1250), and 10 ug/mL DNase I (Thermo Fisher, Cat. no. en0523) dissolved in PBS with 5% Fetal Bovine Serum (FBS; Thermo Fisher, Cat. no. SV30087.02), to digest the SF and skin. The SF and skin tissues were dissociated at 37℃ with a shaking speed of 50 r.p.m for approximately 40 minutes. We collected the dissociated cells at 20-minute intervals to maximize cell yield and viability. Cell suspensions were filtered by the sequence of nylon cell strainer from 100μm to 40 μm, and residual red blood cells were eliminated using 1X Red Blood Cell Lysis Solution (Thermo Fisher, Cat. no. 00-4333-57). The dissociated cells were then washed with 1x DPBS containing 2% FBS. Cell viability was assessed using 0.4% Trypan blue staining and the Countess® II Automated Cell Counter (Thermo Fisher), all samples of suspension cells were filtered by Dead Cell Removal Kit (Miltenyi Biotec, Cat. No. 130-090-101), and only samples with >95 % living cells were used for next steps. Suspension cells were prepared for 10X library construction and 3' sequencing, water-in-oil solution was prepared by the Chromium™ Single Cell Controller (10X Genomics). Beads, each carrying a unique molecular identifier (UMI) and cell barcodes, were loaded to near saturation, ensuring each cell was paired with a bead in a Gel Beads-in-emulsion (GEM). Following exposure to cell lysis buffer, polyadenylated RNA molecules hybridized to the beads. The beads were then collected into a single tube for reverse transcription. During cDNA synthesis, each cDNA molecule was tagged at the 5' end (corresponding to the 3' end of a messenger RNA transcript) with a UMI and a cell label indicating its cell of origin. The 10x beads were then subjected 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 subsequent procedures, including library construction, were performed according to the standard manufacturer's protocol (Chromium SingleCell 3ʹ v3.1). The 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 a NovaSeq6000 (Illumina) using 2x150 chemistry.