GSM9464722: K2_vehicle_cell_hash; Mus musculus; RNA-Seq
Pancretata were harvested from the indicated mouse models and processed for FACS sorting using Collagenase (1 mg/mL) & Dispase (2 U/mL) mix digestion solution and Gentle-MACS dissociator (mTDK_1 protocol), followed by a 0.05% Trypsin-EDTA step. For every sample that would be subjected to cell hashing, we used DNAse-free buffers following digestion. Samples were resuspended in 1 mL ACK-lysis buffer and incubated for 5 min at room temperature to deplete red blood cells, and washed the ACK lysis buffer with 20 mL HBSS. We blocked samples with TruStain Fc block Plus (Biolegend, 156603; clone S17011E, 1:100) for 7 min at 4°C, followed by incubation with a sample-specific TotalSeq cell hashing antibody (Biolegend, 155832, 155833, 155835, 155837, 155839, 155841; clones M1/42; 30-F11). We incubated cell hashing antibodies at a 1:50 dilution for 30 min. We washed samples 3 times with DNAse-free FACS buffer + STI, followed by filtering through a 40-μm strainer and FACS-based isolation of mKate2+/GFP+ cells (KrasG12D+ pancreatic epithelial cells expressing shp53 or shCtrl). To prepare cells for scRNA-seq, we pooled samples from the same experimental conditions into the same tube, and resuspended cells at a 1000 cells μL-1. Cells were resuspended in 1X PBS and BSA (0.04%) and checked for viability using 0.2% (w/v) Trypan Blue staining (Countess II). All sequencing experiments were performed on samples with a minimum of 80% viable cells. Single-cell encapsulation and scRNA-seq library prep of FACS-sorted cell suspensions was performed on the Chromium instrument (10x Genomics) following the user manual (Reagent Kit 3' v3). Each sample loaded onto the cartridge contained approximately 15,000 cells at a final dilution of ~500 cells μl-1. Transcriptomes of encapsulated cells were barcoded during reverse transcription and the resulting cDNA was purified with DynaBeads, followed by amplification per the user manual. Next, the PCR-amplified product was fragmented, A-tailed, purified with 1.2X SPRI beads, ligated to sequencing adapters and indexed by PCR. Indexed DNA libraries were double-size purified (0.6–0.8X) with SPRI beads and sequenced on an Illumina sequencer (R1 – 26 cycles, i7 – 8 cycles, R2 – 70 cycles or higher) to a depth of >50 million reads per sample (>13,000 reads per cell) at MSK's Integrated Genomics Operation Core Facility.