GSM6199223: HRPC-AGEs, rep 1, RNA-seq; Homo sapiens; RNA-Seq
GSM6199223
21 weeks old wide type (wt) and leptin receptor-deficient (db/db) in C57BLKs/J background mice were sacrificed. Kidneys were perfused with PBS, followed by dynabead perfusion. Minced kidneys were digested with 1 mg/ml collagenase II , 1 mg/ml Protease E  plus 50 units/ml DNase I  at 37 °C for 5 min. Separated glomeruli were washed three times, then placed in 2 ml digestion buffer and incubated for an additional 30 minutes at 37℃ with rotating. Globes were removed and dissected in PBS on ice. Retina tissues were dissociated with dissociation system containing 2 mg/ml collagenase  II  and 200 units/ml DNase I at 37℃ for 30 minutes on a rotator. The retinal cells were then sorted by magnetic bead separation using PE anti-mouse CD140b antibody and PE-anti PECAM1 antibody. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. RNA libraries for RNA-seq were prepared using SMARTER mRNA-Seq Library Prep Kit following manufacturer's protocols.