GSM9697974: Mouse ureter, P0, control; Mus musculus; RNA-Seq
Unfixed flash-frozen ureter tissue isolated from 13 control and 14 Pax2-cre/+;DNp63fl/fl;R26mTmG/+ (DNp63cKO) mice at postnatal day (P)0, was sent to Singleron Biotechnologies (Cologne, Germany) for further processing. At Singleron, the tissue samples were thawed and pooled according to their genotype. A single nucleus suspension was obtained for each tissue pool using the GEXSCOPE® Single Nucleus RNA Library Kit V3 (Singleron Biotechnologies). Briefly, the tissue was immersed in cold nucleus separation solution (Singleron Biotechnologies) and cut into small pieces, while kept on ice. Further homogenization was performed using a Kimble Dounce Tissue Grinder (KIMBLE® KONTES® Dounce Tissue Grinder, # 885300-0002). The sample was then incubated on ice for 15 minutes and filtered through a 40-µm sterile strainer. The nuclei were pelleted by centrifugation and their quality was assessed by Trypan Blue staining (Gibco 0.4% w/v Trypan Blue solution, # 11538886, ThermoFisher Scientific, Waltham, MA, USA) under a light microscope. The nuclei were counted using Acridine Orange/Propidium Iodide staining with a Luna FX7 automated cell counter (Logos Biosystems, Villeneuve d'Ascq, France). The concentration of the nucleus suspension was adjusted to 500,000 nuclei/ml. The suspension was then loaded onto a microfluidic chip from the GEXSCOPE™ Single Nucleus RNA-seq Library Kit (Singleron Biotechnologies) to capture approximately 10,000 nuclei in microwells. Transcriptome tagging was performed by adding paramagnetic beads conjugated with oligonucleotide barcodes and unique molecular identifiers (UMIs). Library construction was carried out according to the manufacturer's instructions.