GSM9738019: Hashtag oligonucleotide feature barcode sequencing for multiplexed WT lung, Prf1-/- lung, and Prf1-/- bone samples; Mus musculus; OTHER
Lungs and knees were digested in 1 mL of digestion buffer (2 mL Collagenase I [7.5 mg/mL], 1.49 mg DNase I, 8 mL 1% BSA in HBSS++) per mouse. For lung tissue, tumors were resected and digested in 10 mL of digestion buffer for 20 minutes at 37°C with rotation. After digestion, the material was strained, washed with 5 mL of SMEM + 2% FBS, and resuspended in 1 mL of SMEM + 2% FBS. Red blood cells were lysed with 10 mL of 1× RBC Lysis for 1 minute, neutralized with 10 mL of SMEM + 2% FBS, and strained again. Cells were Fc-blocked (1:200)  (Clone 2.4G2, cat #70-0161) for 10 minutes at 4°C, followed by staining with anti-CD45-PerCPCy5 (1:200) (Clone 30-F11, cat #103132)  and DAPI. Finally, cells were resuspended in 1 mL of PBS + 2% FBS for sorting. For knee tissue, the bones were cleaned, crushed in 1 mL of digestion buffer, and transferred to precoated Eppendorf tubes. An additional 1 mL of digestion buffer was added, and the samples were incubated at 37 °C for 30 minutes, with gentle pipetting every 10 minutes to facilitate digestion. Cells were processed following the same procedure as the lung samples. Both lung and knee tissue samples were subjected to FACS on a BD FACSAria to isolate GFP+CD45-DAPI- cancer cells. B16F10 MTCs extracted from wild type lung, Prf1-/- lung, and Prf1-/- bone were hashed using oligonucleotide-conjugated antibodies (TotalSeq B, hashes 1, 2, and 3) to uniquely label individual samples, which were then pooled together in equal proportions to enable multiplexed scRNA-seq. The pooled cells were processed using the 10x Genomics Chromium platform to generate single-cell gel bead-in-emulsions (GEMs). Reverse transcription and cDNA amplification were performed according to the 10x Genomics 3' v3 protocol. Separate libraries were generated for the gene expression and hashing data. Libraries were sequenced on an Illumina platform to achieve a minimum of 50,000 reads per cell for gene expression and 5,000 reads per cell for hashing.