GSM4654220: single-nuclei data from whole kidney (middle slice) control mouse_rep2; Mus musculus; RNA-Seq
After mice were sacrificed, kidneys were immediately put in ice-cold PBS. A middle slice (1-2mm) containing cortex, outer and inner medulla was then obtained. All samples were then put in 4°C-cold RNAlater for 24 hours and then to -80°C. For the generation of the single-nuclei suspensions (all reactions at 4°C), specimens were thoroughly minced in nuclear lysis buffer 1 (nuclear lysis buffer (Sigma) + Ribolock (1U/µl) + VRC (10mM)) and homogenated using a dounce homogenizer with pastel A (Sigma D8938-1SET), filtered (100µm), homogenized again (douncer with pastel B), filtered through a 35µm strainer and centrifuged (5min, 500g). The pellet was then resuspended in nuclear lysis buffer 2 (nuclear lysis buffer + Ribolock (1U/µl)). To remove debris from the suspension, we underlayed the suspension with lysis buffer containing 10% sucrose and 1U/µl of Ribolock. After centrifugation (5min, 500g), the supernatant and debris were carefully removed. Pelleted nuclei were resuspended in PBS/0.04%BSA + Ribolock (1U/µl), filtered through a 20µm strainer and stained with DAPI. The nuclei suspension was then FACS-sorted (BD FACS Aria II), counted after FACS-sorting and subjected to single-cell sequencing. Library construction followed the exact protocol from 10xgenomics v2 chemistry for 3° scRNA-seq.