Mitochondrial RNA-seq and total RNA-seq of 293T, HeLa, and N2a cells; RIP-seq of SUPV3L1, ceSUPV3L1, ATP5B, and TRAP1; Small RNA-seq of 293T mitochondrial fractions

The mechanism of mecciRNA degradation remains unknown. To investigate the degradation of mecciRNAs, we performed mitochondrial RNA sequencing and total RNA sequencing of 293T, HeLa, and N2a cells. To investigate the degradation mechanism of mecciRNAs, RIP-seq was conducted in 293T, HL-1 cells, and C. elegans. Small RNA sequencing of mitochondrial sucrose gradient fractions was performed to identify mecciRNA degradation fragments. Overall design: 293T, HeLa, and N2a cells were treated with Actinomycin D (ActD) for the indicated time. Mitochondria were isolated from ActD-treated cells and control (DMSO) cells. Mitochondrial RNAs and total RNAs were isolated for ribosomal RNA (rRNA) depleted RNA sequencing (RNA-seq). SUPV3L1 RIP-seq was performed using 293T whole cell and 293T mitochondrial materials. TRAP1 RIP-seq was performed using mitochodnrial fractions of 293T and HL-1 cells. ATP5B RIP-seq was performed using 293T mitochondrial materials. Mitochondria of C. elegans (young adult) were isolated for ceSUPV3L1 and ceELAC2 RNA-seq. RNA of mitochondrial fractions from a 5%–18% sucrose gradient were separated in a 5% urea PAGE gel, and the small RNA bands in fractions were cut off and purified for small RNA sequencing.