GSM8390473: WT1 scRNAseq; Mus musculus; RNA-Seq
Two pooled samples, each comprised of choroids from 1 male and 1 female WT mouse were prepared using a modified version of our previously published protocol for preparation of samples from the anterior segment (Thomson and Quaggin, 2022). Briefly, mice were euthanized before eyes were enucleated and choroids dissected in ice-cold HBSS (Gibco-Life Technologies, Carlsbad CA) under a dissecting microscope. After dissection, pooled choroids were minced and immediately transferred to ice cold DMEM (Gibco) containing 10% fetal bovine serum. Samples were then digested for 2h in 1mg/ml Collagenase A (Roche, Basel, Switzerland) solution containing 1 μM of Y27362 (Roche), washed and digested for an additional 30 minutes in 0.25% trypsin (Gibco) containing 100 units/ml DNAse 1 (Roche). Samples were then washed a second time, passed through a 40 μm filter and submitted to the NU Seq core at Northwestern University for single cell library preparation. Ten thousand cells for each group were loaded into the Chromium Controller (10X Genomics) on a Chromium Next GEM Chip G (10X Genomics), and processed to generate single cell gel beads in the emulsion (GEM) according to the manufacturer's protocol. The cDNA and library were generated using the Chromium Next GEM Single Cell 3′ Reagent Kits v3.1 (10X Genomics) and Single Index Kit T Set A (10X Genomics) according to the manufacturer's manual. Quality control for constructed library was performed by Agilent Bioanalyzer High Sensitivity DNA kit (Agilent Technologies) and Qubit DNA HS assay kit for qualitative and quantitative analysis, respectively.