GSM8142165: 6_Pod_RARRES1_WT; Mus musculus; RNA-Seq
Decapsulated whole kidneys were minced and dissociated in 2 mL of digestion buffer at 37°C for 17 min. The digested tissue was gently resuspended in RPMI medium supplemented with 10% FBS and filtered through a 70-micron cell strainer (Falcon) on ice and pelleted by centrifugation (200g for 5 min), and repeated. The pellet was resuspended in HBSS and further filtered through a 40-micron cell strainer (Falcon) to obtain single cells. After centrifugation, the cell pellets were incubated with 5 mL of RBC lysis buffer (eBioscience, 00-4333-57) to remove any remaining red blood cells on ice for 5 min. Finally, the cells were washed and resuspended in RPMI medium twice to remove any debris or cell aggregates. Cell viability and number were determined using a Countess II Automated Cell Counter (Invitrogen, C10227) after staining with trypan blue. 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.