GSM9365527: Pre-tumor p53 proficient 0; 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. Cell suspensions were incubated with Fc block and CD45-APC or CD45-APC-Cy7 antibody to mark immune cells. Live pancreatic epithelial cells (marked as mKate2-positive, CD45-negative and DAPI-negative) or immune cells (marked as mKate2-negative, CD45-positive and DAPI-negative) were sorted using Aria3 or Aria 1 sorter, and collected in 2% 1x PBS buffer containing 2% fetal bovine serum (FBS). Single-cell suspensions were spun down and resuspended in 0.05% BSA containing 1 U/µL Invitrogen Ambion RNase Inhibitor ​​(AM2682, Thermo Fisher Scientific). 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' v2 or v3). For pre-tumor p53 deficient samples, we spiked-in CD45+ cells to the cell suspension to allow visualization of cell pellets during sample preparation prior to encapsulation. Each sample loaded onto the cartridge contained approximately 5,000 cells (non-hashed samples) or 15,000 cells (hashed samples) 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.