RPL3L-containing ribosomes modulate mitochondrial activity in the mammalian heart.
Here, we explore the biological function of RPL3L, a ribosomal protein (RP) paralog of RPL3L that is exclusively expressed in muscle and heart tissues, by generating a viable homozygous Rpl3l knockout mouse strain. Single nuclei from wild type and Rpl3l knockout mouse strains were obtained from flash-frozen tissues that were dissociated following a previously described method. Tissue homogenization was performed using a 7 mL glass Dounce tissue grinder set (Merck) with 8 strokes of a loose and a tight pestle in homogenization buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl, 1 mM dithiothreitol (DTT), 1X protease inhibitor, 0.4 U µL-1 RNaseIn, 0.2 U µL-1 SUPERaseIn, 0.1% Triton X-100 in nuclease-free water). Homogenate was filtered into a 50 mL tube through a 40-µm cell strainer (Corning) and centrifuged (500 g, 5 min, 4?°C) to resuspend the pellet in 500 µL of storage buffer (1× PBS, 4% bovine serum albumin (BSA), 0.2 U µl-1 Protector RNaseIn). Nuclei were stained with NucBlue Live ReadyProbes Reagents (ThermoFisher) and single nuclei were sorted by fluorescent activated cell sorting (FACS). The obtained tube was centrifuged (500 g, 5 min, 4?°C) to get nuclei pellet. Next, 5 µL of single nuclei suspension were mixed with 5µL of Trypan Blue and applied to a Countess II to determine the nuclei concentration. The suspension was adjusted to 800–1,400 cells per µL and loaded to the Chip, targeting the recovery of 5,000 nuclei per sample. The Chip was processed using the Chromium Controller protocol (10X Genomics). Libraries were prepared following the Chromium Single Cell 3' Reagent Kits User Guide (10X Genomics) protocol. Libraries were sequenced using NovaSeq 6000 flowcell (Illumina). Sequenced single nuclei samples were demultiplexed using bcl2fastq (Illumina) and BAM files were generated by aligning these reads to the annotated mouse reference genome (mm38, Ensembl v98) with CellRanger (v5.0.0), using default parameters and including introns to globally capture expression of pre-mRNA transcripts. Genomic regions corresponding to annotated ribosomal protein pseudogenes were masked out.