GSM5013445: ADX_Female; Mus musculus; RNA-Seq
The corpus from three mice tissue was combined in the same tube and washed 2x in Hanks Balanced Salt Solution without Ca2+ or Mg2+ containing 5 mM HEPES, 5 mM EDTA, and 5% FBS for 20 minutes at 37oC. Tissue was then washed in Hanks Balanced Salt Solution with Ca2+ and Mg2+ for 10 minutes at 37oC and then digested in 1 mg/ml type 1 collagenase (Worthington Biochemical) and 1.5 mg/ml DNase I (Worthington Biochemical, Lakewood, NJ) for 20 minutes at 37oC. After digestions the tissue fragments were pushed through a 100 µm strainer. Fc receptors were blocked with TruStain (Bioledgend, San Diego, CA) and then stained for with anti-CD45 (Cone 104) for 20 minuets at 4oC. Actinomycin D (ThermoFisher) was used to label dead cells. Viable CD45+ cells were sorted using a FACS Aria II (BD Bioscience, San Jose, CA) running FACS Diva software and resuspended at a concentration of 1.0 x 106 cells/ml in RPMI 1640 + 10% FBS. Single cell libraries were prepared on a 10x Genomics Chromium controller (10x Genomics, Pleasanton, CA) using the version 3 reagent kit according to the manufacturer's instructions. The reagent mixture was calculated for capturing 6000 cells. The libraries were sequenced on a NovaSeq (Illumina, San Diego, CA) by the NIEHS Epigenomics core laboratory. Raw read processing was carried out using the Cell Ranger Single-Cell Software Suite (version 3.0.1, 10X Genomics).