GSM5706417: 9_4_Cre; Mus musculus; RNA-Seq
The left kidney was decapsulated, cut in several pieces and 0.15-0.20g of kidney tissue were put in a GentleMACs C tube with with dissociation buffer and were dissociated using the Multi-tissue dissociation kit 1 (Miltenyi, 130-110-201) (Composition of dissociation buffer: 2.35ml serum-free RPMI culture medium (Gibco), 100μl enzyme D, 50μl enzyme R and 12.5μl enzyme A). An initial round of dissociation in the GentleMACS dissociator (“heart_01_01” programme (15s)) was followed by 30 minute incubation in a waterbath (37 oC) which was followed by another round of dissociation (“heart_01_02” programme, 30s). When the second round of dissociation finished, 8ml of sterile PBS/10% FBS were added to the C tubes to stop the reaction. The samples were passed through a 40μm cell strainer into a 50ml falcon tube. The samples were washed with PBS, spun down and resuspended in 1ml red blood cell lysis buffer (8.29g NH4Cl, 1g KHCO3 and 37.2g Na2EDTA in PBS) for 30 seconds. This was followed by incubation with debris removal solution (Miltenyi) and eventually the cells were resuspended in 10ml cold PBS/10% FBS/2.5mM EDTA. Cell viability and concentration were determined using the Trypan Blue assay. 20,000- 40,000 cells were loaded on a 10x Chromium chip (1 sample per lane). Cleanup, reverse transcription, cDNA amplification and library preparation were performed using the Chromium Single cell 3' Reagent Kits (v3) as per the manufacturer's instructions Droplet-based scRNA-seq (Chromium 10x chip)