GSM4856234: 298s_H3; Mus musculus; RNA-Seq
Single cell preparation consisted of two main procedures: (1) Isolation of the glomerulus: mice were first perfused with Dynabeads via heart; minced fresh human healthy biopies and mouse kidney were incubated with Collagenase IV (1mg/ml) and DNase I (50U/ml) at 37°C for 15 and 30 min, respectively. Human samples then were sieved once through 300µm-strainers and isolated glomeruli collected via 100µm-strainers. Mouse samples went through 100µm-strainers twice and bead-containing glomeruli were separated from tubules via a magnet holder followed by 3x wash. (2) Single cell dissociation: isolated human or mouse glomeruli were digested with 1x PBS with Ca++/Mg++ containing collagenase IV (1mg/ml), pronase (1mg/ml) and DNaseI (50U/ml) at 37°C for 15 min with mild shaking (400rpm). Dissociated cells were washed once. For sorting viable single cells, a live cell dye (CMFDA-Green or DRAQ5) was used. The reverse-transcribed cDNA from single cells was amplified 23 cycles followed by tagmentation and a standard index PCR. The single cell libraries were prepared according to Smart-seq2 protocol. The sequencing was performed on HISEQ 3000 (IIlumina).