GSM6757774: nuc2, snRNAseq; Mus musculus; OTHER
The RNALater reserved frozen tissue of 3 mice kidney quarters were thawed and transferred to one petri dish preloaded with 1 ml TST buffer containing 10 mM Tris, 146 mM NaCl, 1 mM CaCl2, 21 mM MgCl2, 0.03% Tween-20 (Roche, 11332465001) and 0.01% BSA (Sigma, B8667). It was minced with a razor blade for 10 minutes on ice. The homogenized tissue was then passed through a 40 µm cell strainer (VWR, 21008-949) into a 50 ml conical tube. One ml TST buffer was used to rinse the petri dish and collect the remaining tissue into the same tube. It was then mixed with 3 ml of ST buffer containing 10 mM Tris, 146 mM NaCl, 1 mM CaCl2 and 21 mM MgCl2 and spun down at 500g for 5 min at 4°C followed by supernatant removal. In the second experiment this washing step was repeated 2 more times. The pellet was resuspended in 100 µl ST buffer and passed through a 35 µm filter. The nuclei concentration was measured using the K2 Cellometer (Nexcelom Bioscience) with the AO nuclei stain (Nexcelom Bioscience, CS1-0108-5ML). 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.