GSM6743945: kidney, TG, day28 [TG_10988]; Mus musculus; RNA-Seq
Mice were perfused with ice-cold PBS and kidney halves collected for snRNA-seq analysis. Half kidney was cut into small pieces (>25) and transferred into a 2-ml Dounce homogenizer (Sigma, Cat#D8938) loaded with 1 ml of NEZ Lysis Buffer (Sigma, Cat#N3408) with RNase inhibitor (NEB, Cat#M0314) at final concentration of 0.4U/µl) on ice. Samples were then Dounce homogenized on ice with five strokes of the looser pestle every 2 min for 8 min (25 strokes in total). Samples were then slowly Dounce homogenized 25 times with the tighter pestle on ice. The  homogenized sample was filtered through a 40-μm Falcon Nylon Cell Strainer, then the filter was washed with 8 ml of 1% BSA PBS, and the nuclear suspension spun in a precooled (4 °C) centrifuge at 650g for 8 min. Supernatant was removed and the pellet resuspended in 1% BSA PBS (plus RNase inhibitor) and moved to a low-bind Eppendorf tube. Nuclear quality and number were assessed with trypan blue staining. Nuclei were sorted as DAPI positive on a BD Aria into 2% BSA solution with RNA inhibitor. Barcoded single-cell gel beads in emulsion (GEMs) were created by 10x Genomics Chromium TM and then reverse transcribed to generate single-cell RNA-seq libraries using Chromium Single Cell 3' Library and Gel Bead Kit v2 (10X Genomics) according to manufacturer's instructions