title: GSM7116302: Glp1r, replicate 1, scRNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: Single cell suspensions were prepared from coronal sections of hindbrain tissue pooled from four to six GiprEYFP or Glp1rEYFP male mice that were four to six weeks old as described previously (1). Tissue from the brain stem located between -6.00 to -8.12 mm relative to Bregma was dissected into Hibernate-A medium without calcium (BrainBits). The tissue was digested with 20 U/ml Papain (Worthington) for 30 min at 37°C, followed by trituration in Hibernate-A medium (Thermo Fisher Scientific) containing 0.005% (w/v) DNase 1 (Worthington). The cell suspension was filtered through a 40 μm cell strainer into a fresh tube.   Fluorescence-activated cell sorting was performed using a BD Influx Cell Sorter (BD Biosciences, Franklin Lakes, NJ, USA) to isolate GiprEYFP+ and Glp1rEYFP+ cells prepared from GiprEYFP or Glp1rEYFP mice, respectively. Cells were gated according to cell size (FSC), cell granularity (SSC), FSC pulse-width for singlets, fluorescence at 488 nm/532 nm for EYFP and 647/670 nm for nuclear stain with DraQ5 (Biostatus). Single cell cDNA libraries from purified GiprEYFP+ or Glp1rEYFP+ cells were generated using the 10× Genomics Chromium Instrument and single-cell 3' Reagent kit (V3; 10X Genomics). Pooled libraries were sequenced on an Illumina NovaSeq 6000 instrument.