GSM7220529: CTR, scRNAseq; Mus musculus; RNA-Seq
Single cells isolated from kidneys of control and transgenic mice were used for Single-cell RNA sequencing. In brief, kidney tissues from control or transgenic mice were cut into small pieces (~1mm2) on ice, washed with cold PBS, and digested at 37°C with a shaking speed of 150 rpm for 40 min in an air bath shaker. The digestion medium containing 450 units/ml collagenase1,  150 units/ml collagenase 2, 450 units/ml collagenase 3, 450 units/ml collagenase 4, 0.8 units/ml Elastase, 300 units/ml Hyaluronidase, 250 units/ml DNase I, and 1 units/ml Dispase. The digested cells were mixed with cold RPMI 1640 with 2% FBS, filtered through a prewetted 70-μm & 40 µm strainer and  centrifuged at 1500 rpm at 4°C for 5 minutes. The cell pellets were then treated for ACK lysis for 10 min, washed with ice-cold PBS+0.04%BSA, and resuspended in cold PBS+0.04%BSA. The prepared cells were then be resuspended for evaluating cell viability, adjusting cell number, and preparing cell library. Single-cell library was prepared using the 10x Genomics Chromium Single Cell 5′ Library & Gel Bead Kit v2 (PN-1000014). Cell suspensions were loaded onto a Chromium Single-Cell Chip with the reverse transcription master mix and single cell 5′ gel beads, aiming for 10,000 cells per channel. Reverse transcription was performed using a C1000 Touch Thermal Cycler (Bio-Rad Laboratories). Quality control and quantification were performed using the Agilent 2100 Bioanalyzer with a DNA high-sensitivity chip. For input into the gene-expression library construction, 50 ng cDNA and 14 cycles were used. Libraries were sequenced using an Illumina HiSeq 2500 NexGen Sequencer