title: NextSeq 500 paired end sequencing; scRNAseq libraries of pooled kidney samples from wild type (ZF1) versus CRISPR-targeted renin knockout (ZF2) zebrafish
design: scRNAseq libraries of pooled kidney samples from wild type (ZF1) versus CRISPR-targeted renin knockout (ZF2) zebrafish
library_construction_protocol: Mesonephric kidneys were isolated into ice-cold Leibowitz medium. Cells were dissociated in medium supplemented with cold activated protease (20mg/ml), DNAse (400units/ml), Liberase (0.085mg/ml), collagenase (2mg/ml) and CaCl2 (5mM), at 6oC for 20 mins with frequent triturition. Samples were transferred to 28.5oC for an additional 10 mins to complete digestion. Digested kidneys were passed through a 40micron sieve prior to centrifugation. Cells were resuspended in PBS with 2% FCS for FAC sorting. A WIK mesonephric kidney was used to control for autofluorescence, and DAPI was used for the live/dead cell count. Samples were sorted for red and green fluorescence, in order to sort renin-expressing cells and smooth muscle cells respectively.  Zebrafish were terminally anaesthetised (tricaine methanesulphonate; MS-222) and decapitated. Lower abdomen was removed to expose kidney which was carefully teased away from the spine. Zebrafish were maintained in a Home Office approved establishment at 28.5oC under standard feeding and light regimen. Renin gene was targetted using CRISPR Cas9 technology. Briefly, an sgRNA,  specifically targeting exon 2 of the renin gene, was complexed with tracr-RNA and Cas9 protein and injected into a one-cell embryo. Surviving fish were genotyped by tail clip and targeted fish were crossed onto WIKs. The genotype of F1 progeny was determined by sequencing across the target site and fish heterozygous for an 8bp deletion in renin exon 2 were crossed to generate ren-/- knockout fish. Nucleic acid was extracted using the Chromium Single Cell 3' Library and Gel Bead kit v2 (10X Genomics, PN-120237) and the Chromium Single Cell A Chip kit (10X Genomics, PN-120236) according to manufacturers instructions. Nucleic acid library was constructed using the Chromium Single Cell 3' Library and Gel Bead kit v2 (10X Genomics, PN-120237) and the Chromium Single Cell A Chip kit (10X Genomics, PN-120236) according to manufacturers instructions.