GSE192419
C. elegans L4 hypodermal SLAM-ITseq
To adapt SLAM-ITseq for Caenorhabditis elegans, we inserted a codon-optimized version of the Toxoplasma gondii UPRT gene downstream of a FRT-flanked mCh::his-58 cassette and under control of the strong and ubiquitous eft-3 promoter. We introduced a single copy of this construct into the C. elegans genome and crossed the resulting line with a dpy-7p::FLP driver. FLP-dependent expression of UPRT was confirmed by Western blotting (strain BN1190). As a proof of concept, we exposed animals to 4-TU from L3 to L4 stage and purified total RNA. RNA from animals without FLP expression was used to control for UPRT-independent labeling strain BN1158). RNA was alkylated with iodoacetamide, which leads to a T>C conversion of 4-TU-labeled positions during cDNA synthesis, and subjected to deep sequencing (see Materials and Methods). We confirmed that the presence of FLP and UPRT did not affect gene expression. Comparing the T>C conversion rate per gene revealed a significant increase in nematodes expressing UPRT in the hypodermis. Next, we performed a beta-binominal test to identify genes that were significantly labelled across all replicas, which retrieved 197 genes (p<0.003; FDR<0.1 after adjustment for multiple comparisons). Original SLAM-ITseq reference: Matsushima W, Herzog VA, Neumann T, Gapp K, Zuber J, Ameres SL, Miska EA (2018) SLAM-ITseq: sequencing cell type-specific transcriptomes without cell sorting. Development 145 (13). doi:10.1242/dev.164640 Overall design: Four replicas of control strain BN1158 (- FLP) and three replicas of strain BN1190 (+ FLP in hypodermis).