GSM8889112: B6.129S4 Strain, Urethane treated, #33; Mus musculus; RNA-Seq
Fresh lung and tumor samples were collected from mice, washed with PBS, and minced into ~1 mm³ pieces in RPMI-1640 medium (Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS, Gibco). The tissues were enzymatically digested with a solution of 1 mg/ml collagenase A, 0.4 mg/ml hyaluronidase, and BSA fraction V (1:5) for 1 ~ 2 hours at 37°C on a rotor, following the manufacturer's instructions.  The digested samples were centrifuged at 350g for 5 minutes, the supernatant was removed, and 1 ~ 5 ml of pre-warmed Trypsin-EDTA was added. The cells were resuspended and gently pipetted for 1–3 minutes, resulting in a stringy consistency due to the lysis of dead cells and released DNA. After adding 10 ml of cold RPMI-1640 without phenol red supplemented with 2% FBS, the samples were centrifuged again at 350g for 5 minutes, and the supernatant was discarded.  If cell clumps persisted, 5 ml of pre-warmed Dispase (5 U/mL) and 50 µL of DNase I solution (10 mg/ml in 0.15 M NaCl) were added, followed by 1 minute of pipetting. For persistent stringiness, an additional 50 µL of DNase I was used. The dissociated cells were diluted with 10 ml of cold RPMI-1640 without phenol red (2% FBS), filtered through a 40 µm cell strainer, and centrifuged at 450g for 5 minutes.  To remove red blood cell contamination, the cell pellet was treated with a 1:4 mixture of cold RPMI-1640 (2% FBS) and ammonium chloride solution, centrifuged, and resuspended in PBS with 1% BSA. The final cell suspension, with viability ≥70% and a density of 7 × 105 to 1.2 × 106 cells/ml, was loaded onto the Chromium Single Cell Controller (10x Genomics) for single-cell gel bead-in-emulsion generation.  scRNA-seq libraries were prepared using the Single Cell 3′ Library and Gel Bead Kit v3.1 and sequenced on the NovaSeq 6000 platform (Illumina). 5ʹ Gene Expression (GEX) Library Construction 5.1 GEX Fragmentation, End Repair & A-tailing 5.2 GEX Post Fragmentation, End Repair & A-tailing Double Sided Size Selection – SPRIselect 5.3 GEX Adaptor Ligation 5.4 GEX Post Ligation Cleanup – SPRIselect 5.5 GEX Sample Index PCR 5.6 GEX Post Sample Index Double Sided Size Selection – SPRIselect 5.7 GEX Post Library Construction QC