GSM8843978: Whole Ear Skin 1; Mus musculus; RNA-Seq
Mice were euthanized, and their ears were excised. Ears was then split into ventral and dorsal sheets and placed in 1.5 ml of Liberase TL (0.4 mg/mL in RPMI; Sigma Aldrich) and 50ug of DNase I (Sigma Aldrich) per ear. Ears were minced and incubated with agitation for 1h at 37°C. Tissue digestion was quenched by adding complete media (RPMI 1640 supplemented with 10% fetal calf serum, Lonza), followed by mechanical disaggregation using program C on the GentleMACS dissociator (Miltenyi Biotec). Insoluble material was removed by filtration, and the pellet was washed twice with 2% neonatal calf serum in Hank's balanced-salt solution (HBSS). Single cell suspensions were stained with Ghost Dye UV 450 or Violet 510 dead cell stain for 405 nm excitation (Tonbo Biosciences) at a 1:1000 dilution in PBS for 30 minutes at 4°C, followed by a wash with 2% NCS in HBSS.The cells were washed, filtered, and resuspended in FACS buffer containing 100μg/mL of DNase I. Live cells were collected using a FACSAria fusion (BD Biosciences) and single cell suspension were used for library construction. Single-cell suspensions were submitted to the Cornell BRC Genomics Facility for processing and sequencing. The Chromium Next GEM Single Cell 3' reagent kit v3 from 10X Genomics® protocol was used for FACS-sorted cell sequencing. A target of 10000 cells across runs were loaded for Gel Beads-in-emulsion (GEM) generation. According to the kit protocol first GEMs were generated, subsequently reverse transcription was performed, and cDNA was cleaned up, amplified, and size selected. Sample quality was confirmed using a Qubit (RNA HS kit; Thermo Fisher) to determine concentration and a Fragment Analyzer (Agilent) to determine RNA integrity. The libraries were sequenced with an Illumina NextSeq 2000.