title: GSM4273672: Pancreatitis non-tuft epithelial cells-5; Mus musculus; RNA-Seq
library_construction_protocol: Pancreatic tuft cells were isolated from 5 CD1 male mice with caerulein-induced pancreatitis.  The pancreas was quickly dissected, minced in 5 ml of DMEM with FBS and allowed to incubate at room temperature for 2 min.  Supernatant and fat were removed and pancreatic tissue was then incubated in 10 ml DMEM supplemented with 1 mg/ml collagenase I (Sigma), 1 mg/ml soybean trypsin inhibitor (Gibco), 1.5 mg/ml hyaluronidase (Sigma), and 250 μl of DNAse I, shaking gently at 37°C for a maximum of 30 min.  Digestion was monitored and tissue was further digested mechanically by pipetting.  Digested tissue was passed through a 100 μm filter, washed with FACS buffer (PBS, 1 mM EDTA, 0.5% BSA) containing DNase I, and incubated with ACK lysing buffer (Gibco) to remove red blood cells before staining for FACS. Single cell suspensions were incubated on ice with mouse Fc receptor block (BD Biosciences, 1:200) followed by antigen-specific antibodies in FACS buffer.  DAPI (molecular probes, 1:1000) and Annexin V (Biolegend, Pacific Blue conjugate at 1:200) were used to exclude dead and dying cells.  Cells were labeled with Cd45 (Alexa Fluor 488), EpCAM (Alexa Fluor 647), and Siglec F (PE) (Biolegend, 1:200).  Fluorescence Minus One (FMO) staining controls were included for gating populations of interest. Cells were FACS purified at the Salk Institute's Flow Cytometry core facility on a BD Biosciences Influx cell sorter (100-µm size nozzle, 1 x PBS sheath buffer with sheath pressure set to 20 PSI). Cells were sorted in 1-drop Single Cell sort mode for counting accuracy; these were deposited directly into lysis buffer composed of DNase/RNase-free water, Triton X-100, and Ribolock (Thermo Fischer) in a 96 well plate. Low input bulk RNA sequencing (RNA-seq) on pancreatic tuft cells was performed using the Smart-Seq2 protocol as previously described. In brief, five biological replicates, each with 100 tuft cells from an individual mouse, were sorted directly into 2 μl of Smart-Seq2 lysis buffer. Full-length cDNA was generated, and size distribution was checked with Agilent TapeStation 4200 to ensure RNA quality. cDNA were then amplified with 18-24 PCR cycles, tagmentated, and amplified again with 10 PCR cycles using a Nextera XT kit (Illumina FC-131-1096). The sequencing library was purified with AMPure XP beads. SmartSeq2 (Picelli et al., 2014) or TruSeq stranded mRNA Sample Preparation Kit v2.
attributes.tag: GEO Accession
attributes.value: GSM4273672