title: GSM6514379: VPR1; Mus musculus; RNA-Seq
library_construction_protocol: To induce transgene expression, VPR;Pax8-rtTa mice were fed DOX grain-based rodent diet at 7 week of age and continued for 11 weeks induction until mice were euthanized. The mice were anesthetized with Ketamine/Xylazine and perfused with chilled 1x PBS via the left ventricle. Kidneys were decapsulated, minced into 1-mm3 pieces with a sterile razor blade, and incubated in 2 mL of digestion buffer containing 1 mg/mL collagenase type II (Sigma), 1 mg/mL Pronase E (Sigma), and 50 mg/mL DNase I (Roche) at 37°C for 15 min. The partially digested tissue was then passed through a 70 mm cell strainer (Falcon) into RPMI medium supplemented with 10% FBS twice on ice and pelleted by centrifugation (200 g for 5 min). Then, the pellet was resuspended in HBSS and sheared with a 27G needle to remove large clumps. They were further filtered through a 40 mm cell strainer (Falcon) to obtain a single-cell solution. After centrifugation, the cell pellets were incubated with 3 mL of RBC lysis buffer (eBioscience 00-4333-57) to remove red blood cells on ice for 5 min. Finally, the cells were washed and resuspended in RPMI medium two times to remove debris or cell aggregates. Cell viability and number were determined using a Countess II Automated Cell Counter (Invitrogen, C10227) after staining with trypan blue. By using this method, we were able to generate single-cell suspensions with >80% viability. Sequencing libraries were constructed according to 10x standard protocol by following the steps cDNA fragmentation, end repair and A-tailing, size selection by SPRIselect beads (Beckman Coulter), adaptor ligation, sample index PCR amplification, and repeat SPRIselect beads size selection. The final constructed single-cell libraries were sequenced by Illumina Novaseq machine with total reads per cell targeted for a minimum of 50,000.