GSM4598899: normal-lung_gex; Mus musculus; RNA-Seq
C57BL/6 mice were injected intravenously into the tail vein with 5×10e5 TC1 tumor cells. At 3 weeks post tumor inoculation, tumor-bearing mice were sacrificed. The lungs from tumor-bearing mice and age-matched normal mice were perfused and harvested. Lung tissues were chopped into 1mm3 pieces and digested in RPMI medium supplemented with 10% FBS, 1% P/S, 1% Collagenase IV (Worthington Biochemical corp., Lake wood, NJ, USA), and 0.01% DNase (Sigma Aldrich, St. Louis, MO, USA) for 20 min at 37 °C. The dissociated tissues were filtered through a 40-μm cell strainer. The pulmonary lymphocytes were isolated by conducting a Percoll gradient (Millipore Sigma Corp.) and enriched the CD4+ T cells using a CD4+ T cell isolation kit (Miltenyi Biotec) followed by manufacture's protocol. The enriched CD4+ T cells were stained with the CD4-fluorescent dye-conjugated antibody and excluded the dead cells by staining the Live/Dead Fixable Near-IR Dead Cell Stain Kit (Invitrogen). The single-cell suspensions were sorted into live CD4+ T cells using fluorescence-activated cell sorting (BD Aria III) for further scRNA- and TCR- sequencing analysis. To construct libraries and sequencing, we applied the Chronimum 10x Genomics platform with a V(D)J Enrichment kit and 5' Library & Gel Bead Kit (10x Genomics). To achieve 10,000 targeted cells, the cell suspension was diluted in nuclease-free water and then combined with Single Cell 5′ Gel Beads, a master mix, and Partitioning Oil on Chromium Chip A (GEM). Following to generate the GEMs, RNA transcripts were uniquely barcoded and reverse-transcribed within droplets. 10x Barcoded, full-length cDNA were pooled and enriched via PCR. For V(D)J Enriched Library, the enriched cDNA pool was amplified using primer specific to TCR as in T Cell Mix1 and T Cell Mix2 primer. For 5' Gene Expression (GEX) Library, the cDNA pool went through an end repair process, A-tailing, adaptor ligation, and sample index PCR to create the 5' Gene Expression Library. The purified libraries were quantified and qualified using qPCR Quantification Protocol Guide (KAPA) and the Agilent Technologies 4200 TapeStation (Agilent technologies). Afterwards, the libraries were sequenced using HiSeqX platform (Illumina) according to the read length in the user guide.