Animals were fed five times with unc22 (control) and kiaa1429 dsRNA. Animals were starved for a week following RNAi feedings. Cells were purified for scRNAseq from either control or kiaa1429 (RNAi) animals by FACS using hoechst‐labeling, as previously described (Fincher et al, 2018 (link)). Cells were counted and collected by centrifugation (300 g, 5 min). The supernatant was discarded, and the cells were resuspended in 1× PBS with 0.5% BSA to achieve an optimal cell concentration of approximately 1,000 cells per μl. Cells were counted automatically using the Invitrogen Countess II (Thermo Fisher Scientific), and the cell viability was assessed by labeling with trypan blue (0.4%) and counting positive cells.
Libraries were then prepared at the Genomic Research Unit at Tel Aviv University using the 10× Genomics Chromium Controller in conjunction with the single‐cell 3′ v3.1 kit, protocol revision D. Briefly, cell suspensions were diluted in nuclease‐free water according to the manufacturer's protocol to 10,000 cells in each sample. The cDNA synthesis, barcoding, and library preparation were carried out according to the manufacturer's instructions. cDNA libraries were sequenced on Illumina NextSeq 550 at Tel Aviv University.
Libraries were then prepared at the Genomic Research Unit at Tel Aviv University using the 10× Genomics Chromium Controller in conjunction with the single‐cell 3′ v3.1 kit, protocol revision D. Briefly, cell suspensions were diluted in nuclease‐free water according to the manufacturer's protocol to 10,000 cells in each sample. The cDNA synthesis, barcoding, and library preparation were carried out according to the manufacturer's instructions. cDNA libraries were sequenced on Illumina NextSeq 550 at Tel Aviv University.