GSM6751679
Freshly collected mouse SCLC tumors and allograft tumors were transferred to a dry dish and minced into pieces with blades. The tissue was digested in Leibovitz's medium (Invitrogen) with 2 mg/mL Collagenase Type I (Worthington), 2 mg/mL Elastase (Worthington) and 2 mg/mL DNaseI (Worthington) at 37 °C for 45 min. The tissue was triturated with pipet every 15 min of digestion until homogenous. The digestion was stopped with FBS (Invitrogen) to a final concentration of 20%. The cells were filtered with a 70 μm cell strainer (Falcon) and spun down at 5000 rpm for 1 min. The cell pellet was resuspended in red blood cell lysing buffer (Sigma) for 3 min, spun down at 5000 rpm for 1 min and washed with 1 mL ice-cold Leibovitz's medium with 10% FBS. Cells were resuspended in 1 mL ice-cold Leibovitz's medium with 10% FBS and filtered with a cell strainer (20 μm). Dead cells were removed with Dead Cell Removal Kit (Miltenyi Biotec) according to the manufacturer's instructions. Live cells were collected for 10x Genomics library preparation. Single-cell Gene Expression Library was prepared according to the Chromium Single Cell Gene Expression 3v3.1 kit (10x Genomics). Briefly, single cells, reverse transcription (RT) reagents, Gel Beads containing barcoded oligonucleotides, and oil were loaded on a Chromium controller (10x Genomics) to generate single-cell GEMS (Gel Beads-In-Emulsions), where full-length cDNA was synthesized and barcoded for each single cell. Subsequently, the GEMS were broken and cDNAs from each single cell were pooled, followed by cleanup using Dynabeads MyOne Silane Beads and cDNA amplification by PCR. The amplified product was then fragmented to optimal size before end-repair, A-tailing, and adaptor ligation. The final library was generated by amplification.