title: GSM8519330: Trim66_KO_het_10X_scRNA_seq; Mus musculus; RNA-Seq
library_name: GSM8519330
library_construction_protocol: The MOE tissues were dissected and transported in sterile culture dish with 10 ml 1´Dulbecco's Phosphate-Buffered Saline (DPBS; Thermo Fisher, 14190144) on ice to remove the residual tissue storage solution, then minced on ice. We used the dissociation enzyme, 0.25% Trypsin (Thermo Fisher, 25200-072) and 10 μg/mL DNase I (Sigma, 11284932001) dissolved in PBS with 5% Fetal Bovine Serum (FBS; Thermo Fisher, SV30087.02) to digest the tissues. The MOE tissues were dissociated at 37 °C with a shaking speed of 50 rpm 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 Solution (Thermo Fisher, 00-4333-57). Dissociated cells were washed with 1´DPBS containing 2% FBS. Cells were stained with 0.4% Trypan blue (Thermo Fisher, 14190144) to check the viability on Countess® II Automated Cell Counter (Thermo Fisher). Each of the solutions above contained transcriptional inhibitors (5 μg/mL Actinomycin D., 10 μg/mL Anisomycin, and 10 μM Triptolide, Sigma) 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 (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 (CG000206 Rev D). 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) using 2´150 chemistry.
identifiers.namespace: GEO
identifiers.id: GSM8519330_r1