title: NextSeq 500 sequencing; Analysis of mitochondrial gene expression with Ribosome Profiling
design: Analysis of mitochondrial gene expression with Ribosome Profiling
library_construction_protocol: For Ribo-Seq, parental HEK293 and PDE12-/- cells were treated with 100 μg/ml chloramphenicol and 100 μg/mL cycloheximide for 5 min. Cells were rinsed with 5 ml of ice-cold PBS, the dishes were submerged in a reservoir of liquid nitrogen for 10 s and transferred to dry ice. Cells were lysed in 400 μl of lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1 % Triton X-100, 100 μg/ml chloramphenicol and cycloheximide). Cells were scraped and lysates clarified by centrifugation for 20 min at 13,000 g at 4°C. 200 μl aliquots of cell lysates were treated with 7.5 μl RNase 1 (100 units/μl) at 28°C. The reaction was layered onto a 10-30 % sucrose gradient [2ml 10-30% (v/v)] in 50mM Tris-HCl (pH7.2), 20mM Mg(OAc)2, 80mM NH4Cl, 0.1M KCl, 1mM PMSF and centrifuged for 2h 15min at 100,000 gmax at 4°C (39,000 rpm, Beckman Coulter TLS-55 rotor) and fractions collected. Subsequently, all fractions containing mitochondrial monosomes, ascertained by western blotting, were pooled and digested with proteinase K (200 μg/ml) for 30 min at 42°C. RPFs were recovered by extracting twice with pre-warmed (65°C) acidic phenol:chloroform and once with chloroform (1:1, v/v, buffered with 10 mM Tris pH 7.5, 0.1 mM EDTA) followed by ethanol precipitation. RPFs (1 μg) were separated on 15 % denaturing polyacrylamide gels and RNA species migrating between 25 and 35 nt were harvested. RNA was eluted from the gel slices and ethanol precipitated. The RNA samples from above were heated at 80°C for 2 min, cooled and the 3’ phosphate group removed using T4 polynucleotide kinase (New England Biolabs) for 2 h at 37°C in a 20 μl reaction. The RNA was concentrated by ethanol precipitation, resuspended in 10 mM Tris-HCl (pH 7.5) and ligated in a 20 μl reaction overnight at 14°C to a preadenylated 3’-adaptor using T4 RNA Ligase 2 (New England Biolabs). RNA was precipitated, loaded into a 15 % denaturing polyacrylamide gel and ligated RNA fragments migrating between 48 and 54 nt were excised. The RNA was eluted, precipitated, resuspended as above, and 5’ phosphorylated using T4 PNK in the presence of 1 mM ATP for 2 h at 37°C. RNA was concentrated by ethanol precipitation, resuspended in 10 mM Tris-HCl (pH 7.5) and ligated to a 5’ RNA adaptor overnight at 14°C using T4 RNA Ligase (New England Biolabs). The fully adapted RNAs were recovered by ethanol precipitation, dissolved in 6 μl 10 mM Tris-HCl (pH 7.5), and ligated product annealed to an RT primer for 5 min at 65°C. The RNA was subsequently reverse transcribed for 50 min at 55°C in using SuperScript III according to manufacturer’s instructions, followed by heat inactivation for 5 min at 85°C. Standard PCR reactions were used to prepare amplicons using forward primer RP1 and RPIX as reverse primer, where X is primer number (1-24). PCR reactions were loaded onto 10 % non-denaturing polyacrylamide-TBE gels and products of ~150 bp were excised from the gel and eluted. These amplicon libraries were ethanol precipitated and resuspended in 15 μl 10 mM Tris-HCl (pH 7.5). Ribosomal RNA was depleted from Ribo-seq samples at the library amplicon stage; 12 µL of the relevant Ribo-seq library was mixed with 4 µL of 4× hybridization buffer (200 mM HEPES pH 7.5 and 2 M NaCl) and denatured at 98°C for 2 min. DNA was re-annealed for 5 h at 68°C prior to addition of 2 µL of 10× DSN master buffer and 2 µL of DSN (4 units, Evrogen). Digestion was allowed to proceed for 25 min at 68°C, before addition of 20 µL 10 mM EDTA and incubation for a further 5 min at 68°C. DNA was recovered by a single extraction with phenol–chloroform (1:1, vol/vol) followed by ethanol precipitation and resuspended in 4 µL 10 mM Tris–HCl pH 7.5. The treated amplicon library was subjected to another round of PCR (as above) and the resulting library subjected to a second round of DSN treatment.