GSM7431962: FOXI1-KO ferret airway epithelial cells - Donor 8; Mustela putorius furo; RNA-Seq
Fully differentiated ferret airway epithelia ALI cultures were dissociated using Accumax (Stem Cell Technologies) followed by DNase treatment. Cells were filtered through a 20 μMstrainer and pelleted in 0.04% BSA PBS at 500g for 10 min. Nonviable dead cells were removedby using MACS Dead Cell Removal Kit following 10X Genomics recommendations (DocumentCG00039). Single cells were counted on a Thermo countess cell counter and 0.04% BSA/PBSwas added to achieve a targeted concentration of 1000 cells/μL. Ionocyte-enrichment wasperformed on OH-Tam treated FOXI1-CreERT2::ROSA-TG ALI cultures followed by FACSisolation of EGFP and Tomato positive cells. FACS isolated EGFP positive cells where thenmixed with Tomato positive epithelial cells to achieve ~10,000 total cells for 10x sequencing.The ratio of EGFP to Tomato positive cells varied for each experiment depending on the yield oflineage-labeled cells. Sequencing libraries were generated by following 10X Genomicsrecommendations (Document CG000315). Briefly, single cells and reverse transcription mastermix were partitioned into Gel Beads in partitioning oil in the 10X Chromium controller. Afterreverse transcription, cDNA libraries were amplified and fragmentated, followed by adaptorligation and sample index PCR reaction. Libraries were sequenced on NovaSeq 6000 platform by the University of Iowa Genomics Division.