title: GSM6783893: Diabetic + Vehicle (DV) 1; Mus musculus; RNA-Seq
library_name: GSM6783893
library_construction_protocol: Preparation of kidney tissues was conducted at 4˚C with all buffers maintained on ice. Processed individually, each mouse kidney was collected and chilled in PBS before dissection of kidney cortex. The isolated cortex material was initially minced using a scalpel and pressed through a 100µM cell sieve using a 1 mL syringe plunger while rinsing material through with PBS. The resulting homogenate was collected in a 50 mL falcon tube and centrifuged at 250 x G for 2 minutes and the supernatant discarded. The pellet was resuspended in 20 mL PBS and poured through a 70 µM sieve to capture glomeruli while allowing smaller structures to pass through. The “glomerular fraction" caught by the sieve was transferred to a 15 mL falcon tube using a Pasteur pipette with a volume of PBS up to 15 mL. The recovered fraction was centrifuged at 200 x G for 5 minutes, after which the supernatant was carefully removed to avoid disturbing pelleted material. The glomerular pellet was resuspended in 1 mL of digestion buffer (10 mg protease from bacillus licheniformis, 10uL DNase, 1uL 0.5 M EDTA, 1mL PBS), and allowed to digest for 13 minutes at 4˚ C with slow rotation. Further digestion was stopped by the addition of 1 mL of 20 % FBS in PBS. To aid dissociation, the mixture was additionally triturated using a 1000 µL pipette very gently. Triturated material was passed through a 40 µM cell sieve and collected in a 50 mL falcon tube by rinsing through with 2% FBS in PBS. Material that did not pass through the 40 µM sieve was further triturated until no remaining clumps remained on the sieve. Immediately prior to starting single cell capture, the dissociated glomerular fraction was centrifuged at 200 x G for 5 minutes, followed by repeated washes (at least 3 washes of 200 x G, 1 mL filtered PBS with 0.01% BSA, 2 minutes) to reduce free RNA in solution Single-cell capture and transcriptome profiling were conducted using a droplet based technology “Drop-Seq” and a single-cell microfluidics platform designed by Dolomite-Bio according to “Drop-Seq Protocol version 3.1 Dec-2015” with minor modifications. Modifications include the use of Terra PCR Direct Polymerase Mix for cDNA  amplification steps with 4 cycles in stage one and 11 cycles in stage two). Final sample libraries were created using the Nextera XT DNA Library Preparation Kit and sequenced using Illumina HiSeq in paired-end 150-cycle configuration by NovogeneAIT Genomics (Singapore).