Illumina HiSeq 4000 sequencing; mitoRibo-seq and RNA-seq of parental and MRM2 knockout HEK 293T Flp-In T-REx cells
mitoRibo-seq and RNA-seq of parental and MRM2 knockout HEK 293T Flp-In T-REx cells
Cells were incubated with 200 g mL-1 chloramphenicol for 10 min and 200 g mL-1 cycloheximide for an additional 5 min. After washing with PBS, the monolayer was flash-frozen on liquid nitrogen and thawed in lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 200 g mL-1 chloramphenicol, 200 g mL-1 cycloheximide). Samples are paired, with Samples 1 to 12 are RNA-seq and samples 13-24 are mitoRibo-seq For the Ribo-seq libraries, half of the clarified lysates was incubated with 375 mU L-1 RNase I at 28 C for 30 min, and monosomes recovered by ultracentrifugation in a continuous 10-30% (w/v) sucrose density gradient containing chloramphenicol and cycloheximide. After 40 mU L-1 proteinase K incubation at 42 C for 30 min, ribosome-protected fragments were extracted in acid phenol-chloroform. For the paired RNA-seq libraries, total RNA was extracted from the other half of the clarified lysates using TRIzol LS, DNA degraded using TURBO DNase and mt-rRNA depleted using the Ribo-Zero Gold kit before alkaline fragmentation (45 mM sodium bicarbonate, 5 mM sodium carbonate, 1 mM EDTA, 95 C, 15 min).