GSM9083964: Wdr4^f/f rep2, scRNA; Mus musculus; RNA-Seq
Tissues were transported on ice in sterile culture dishes containing 10 mL of 1× Dulbecco's Phosphate-Buffered Saline (DPBS; Thermo Fisher, Cat. no. 14190144) to remove residual tissue storage solution, and then minced on ice. Tissue digestion was performed using 0.25% Trypsin (Thermo Fisher, Cat. no. 25200-072) and 10 µg/mL DNase I (Sigma, Cat. no. 11284932001), dissolved in PBS supplemented with 5% Fetal Bovine Serum (FBS; Thermo Fisher, Cat. no. SV30087.02). The tissues were incubated at 37°C with shaking at 50 r.p.m. for approximately 40 minutes. Dissociated cells were collected every 20 minutes to improve cell yield and viability. Cell suspensions were passed through a 40 µm nylon cell strainer, and red blood cells were removed using 1× Red Blood Cell Lysis Solution (Thermo Fisher, Cat. no. 00-4333-57). The dissociated cells were then washed with 1× DPBS containing 0.4% FBS. Cell viability was assessed using 0.4% AO/PI staining on a Countstar Rigel S2 system (Countstar, Shanghai). Samples were subsequently submitted to Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China) for downstream processing. Beads containing unique molecular identifiers (UMIs) and cell barcodes were loaded at near-saturation levels to ensure that each cell was encapsulated with a bead in a Gel Beads-in-Emulsion (GEM). Upon exposure to cell lysis buffer, polyadenylated RNA molecules hybridized to the oligonucleotides on the beads. Beads were then recovered into a single tube for reverse transcription. During cDNA synthesis, each cDNA molecule was tagged at the 3′ end (corresponding to the 5′ end of the mRNA transcript) with both a UMI and a cell-specific barcode to indicate its cell of origin. Subsequently, the beads were subjected to second-strand cDNA synthesis, adaptor ligation, and universal amplification. Sequencing libraries were constructed from randomly fragmented whole-transcriptome amplification products, enriching for the 3′ ends of transcripts that were linked to cell barcodes and UMIs. All remaining procedures, including library construction, were performed following the manufacturer's protocol for the Chromium Single Cell 3′ v3.1 platform (10x Genomics). Libraries were quantified using the High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 and the Qubit High Sensitivity DNA Assay (Thermo Fisher Scientific). Sequencing was carried out on Illumina NovaSeq X Plus and MGI DNBSEQ-T7 platforms using 2×150 bp paired-end chemistry. All sequencing was performed at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).