GSM6734826: 11oldEoE; Mus musculus; RNA-Seq
Esophageal epithelium-enriched tissue layer was peeled from muscle then incubated in 1 ml 1X Dispase I (354235; Corning; Bedford, MA, USA) diluted 1:4 in Hank's Balanced Salt Solution (HBSS; 14025-076; Gibco; Grand Island, NY, USA) for 10 minutes at 37 °C with shaking at 1000 RPM (5384; Eppendorf F1.5 Thermomixer; Hamburg, Germany). Following removal from Dispase I, esophageal epithelium was minced with sharp scissors then incubated in 1 ml of 0.25% Trypsin-EDTA (25-053-CI; Corning; Manassas, VA, USA) for 10 min at 37 °C with shaking at 1000 RPM. Trypsin and tissue pieces were forced through 70 μm cell strainer into 50 ml conical tube containing 4 ml soybean trypsin inhibitor (STI; 9035-81-8; Grand Island, NY, USA). Cells were pelleted at 1300 RPM for 3 min then resuspended in 500 μl complete mouse keratinocyte–serum-free medium (37010022; Gibco; Carlsbad, CA, USA). Cell number and viability were measured using Automated Cell Count (AMQAX1000, Invitrogen Countess II, Bothell, WA, USA) by mixing 5 μl cell suspension with 5 μl 0.4% trypan blue solution (T10282; Invitrogen; Eugene, OR, USA). Library was performed according to the manufacter's instructions (Illumina inDrop). In brief, single epithelial cells were isolated into droplets and lysed. Droplets were fused with hydrogel microsphere containing cell-specific barcodes and underwent reverse transcription, with barcodes serving as primers. cDNA was purified from droplets and ligated to adapters, where they were amplified, annealed to indexed primers, and further amplified prior to sequencing.