GSM6757772: rep2, scRNAseq; Mus musculus; RNA-Seq
One of each kidney sagittal quarter from three perfused mice of different strains C57BL/6, CAST/EiJ and 129S1/SvImJ were harvested into cold RPMI (Thermo Fisher Scientific, 11875093) with 2\% heat-inactivated Fetal Bovine Serum (Gibco, Thermo Fisher Scientific, 16140-071; FBS) and 1\% penicillin/streptomycin (Gibco, Thermo Fisher Scientific, 15140122). Each piece of the tissue was then minced for 2 minutes with a razor blade in 0.5 ml 1x liberase TH dissociation medium (10x concentrated solution from Millipore Sigma, 05401135001, reconstituted in DMEM/F12(Gibco, Thermo Fisher Scientific, 11320-033 in a petri dish on ice. The chopped tissue pieces were then pooled into one 1.5 ml Eppendorf tube and incubated in a thermomixer at 37°C for 1 hour at 600rpm with gentle pipetting for trituration every 10 minutes. The digestion mix was then transferred to a 15 ml conical tube and mixed with 10 ml 10\% FBS RPMI. After centrifugation in a swinging bucket rotor at 500g for 5 min at 4°C and supernatant removal, the pellet was resuspended in 1ml red blood cell lysing buffer (Sigma Aldrich, R7757). The suspension was spun down at 500g for 5 min at 4°C followed by supernatant removal. The pellet cleared of the red blood cell ring was then resuspended in 250 µl Accumax (Stemcell Technologies, 7921) and incubated at 37°C for 3 mins. The reaction was stopped by mixing with 5 ml 10\% FBS RPMI and spinning down at 500g for 5 min at 4°C followed by supernatant removal. The cell pellet was then resuspended in PBS with 0.4\% BSA (Sigma, B8667) and passed through a 30 µm filter (Sysmex, 04-004-2326). The cell suspension was then assessed for viability and concentration using the K2 Cellometer (Nexcelom Bioscience) with the AOPIcell stain (Nexcelom Bioscience, CS2-0106-5ML). The cells or nuclei were loaded onto a 10x Chromium Next GEM G chip (10x Genomics, 1000120) aiming for recovery of 10,000 cells or nuclei. The RNA-seq libraries were prepared using the Chromium Next GEM Single Cell 3' Reagent kit v3.1 (10x Genomics, 1000121) following vendor protocols. The libraries were pooled and sequenced on NovaSeq S1 100c flow cells (Illumina) with 28 bases for read1, 55 bases for read2 and 8 bases for index1 and aiming for 20,000 reads per cell.