title: Illumina HiSeq 4000 sequencing; mitoRibo-seq and RNA-seq of parental and MRM2 knockout HEK 293T Flp-In T-REx cells
design: mitoRibo-seq and RNA-seq of parental and MRM2 knockout HEK 293T Flp-In T-REx cells
library_construction_protocol: 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. Both RNA sample sets were subjected to size selection (25 nt to 35 nt) after TBE-Urea PAGE. Following, RNA was extracted from gel pieces (300 mM sodium acetate pH 5.5, 0.25% SDS, 4 C, 18 h) and standard library preparation was carried out using the TruSeq Small RNA Library Preparation kit (Illumina).