GSM9483893: PDE6G; Mus musculus; RNA-Seq
Fresh mouse tumor tissues were harvested and immediately placed in ice-cold HBSS supplemented with Mg²⁺ and 0.04% (w/v) BSA. Under sterile conditions, the tissues were minced into 1–2 mm³ pieces, then resuspended in digestion buffer (HBSS with Mg²⁺, containing 2 mg/ml collagenase IV, 0.2 mg/ml hyaluronidase, and 40 U/ml DNase I) and incubated at 37°C for 45–60 minutes with gentle shaking. Digestion was terminated by adding an equal volume of FACS buffer (HBSS + 2% FBS + 5 mM EDTA), and the resulting suspension was filtered through a 70 μm cell strainer to remove undigested tissue debris, followed by centrifugation at 300×g for 5 minutes at 4°C. The collected cell pellet was washed twice with FACS buffer, and cell viability and concentration were assessed via Trypan Blue staining to ensure a viability rate of >80% and a final cell concentration of 700–1,200 cells/μl in HBSS containing 0.04% BSA. The prepared cell suspension, reaction mixture (Master Mix + Sample), 10X Barcode Gel Beads (VDJ 5' Gel Beads v1.1), and Partitioning Oil were loaded into separate wells of a Chromium Next GEM Chip G. Through the microfluidic “single cross” system, Gel Beads-in-Emulsions (GEMs) were formed. To achieve single-cell resolution, cells were subjected to limited dilution during GEM generation, resulting in 90–99% of GEMs being cell-free and the remaining GEMs typically containing a single cell. All generated GEMs were pooled together, followed by Gel Bead dissolution and cell lysis within each GEM. The Gel Beads consist of a gel matrix and oligonucleotides conjugated to magnetic beads. Upon dissolution, the Gel Beads release a large quantity of primer sequences, which are composed of: (i) Illumina TruSeq Read 1 sequencing primer, (ii) a 16-nt 10X Barcode (unique to each Gel Bead), (iii) a 10-nt unique molecular identifier (UMI), and (iv) a 13-nt Template Switch Oligo (TSO). The released oligonucleotides were mixed with the lysed cells and Master Mix (containing RT reagents and RT primers) to perform reverse transcription, yielding full-length cDNA sequences tagged with the 10X Barcode. After reverse transcription, the oil-in-water emulsion was disrupted using a Recovery Agent. The cDNA was then recovered, purified, and amplified with DNA polymerase. Following cDNA amplification, an aliquot of the cDNA from a single sample was split into 2–3 portions, which were separately used for TCR/BCR enrichment and library construction, as well as 5' transcriptome library construction, according to the experimental design. V(D)J Enrichment and Library Construction: The constant (C) regions of TCR and BCR genes are highly conserved. Nested PCR was performed using outer and inner primers designed against these C regions to specifically enrich cDNA fragments containing the V(D)J regions. The enriched cDNA was fragmented by enzymatic digestion, and fragments of appropriate size were selected. After end repair, A-tailing, and ligation of Read 2 sequencing primers, the cDNA library containing P5 and P7 adapters was constructed via PCR.