NextSeq 500 paired end sequencing; Single-cell RNA-seq (10x Chromium V3) of zebrafish csf1ra and csf1rb mutant whole kidney marrows
Single-cell RNA-seq (10x Chromium V3) of zebrafish csf1ra and csf1rb mutant whole kidney marrows
Whole kidney marrow was collected from 6 months old zebrafish wt, csf1raΔ5bp, or csf1rbΔ4bp mutant zebrafish animals. Kidney marrow tissue was dissociated by pipetting, washed once with PBS and fractionated using Ficoll-Hypaque/Biocoll (density 1.077 g/ml) centrifugation at 500g for 10 minutes to remove unwanted mature erythrocytes and dead cells. Further, total cell concentration and viability were determined using a TC20 Automated Cell Counter (Bio-Rad). Samples were then resuspended in 1XPBS with 10% BSA at a concentration between 800-3000 per ml and loaded on the 10XChromium system. Samples were loaded on the 10x Chromium system with a target of 4,000 cells. Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1, 16 rxns PN-1000121 was used for library preparation. Samples were loaded on the 10x Chromium system with a target of 4,000 cells. Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1, 16 rxns PN-1000121 was used for library preparation.