Bio spin p30 columns

Misaminoacylated [³²P]tRNAs were prepared by mixing 25 μM tRNA^Ile with 5 μM T243R/D342A IleRS (mutant inactive in post-transfer editing) and a particular amino acid at the following concentration (4 mM Ala or Met; 2 mM Val, Nle or Thr; 0.2 mM Leu; 10 mM Ser) in a buffer containing 20 mM HEPES pH 7.5, 10 mM MgCl₂, 150 mM NH₄Cl, 2 mM ATP, 0.008 U/μl TIPP, 0.01 mg/ml BSA (New England Biolabs). The main purpose of the TIPP is shifting the equilibrium of aminoacylation reaction towards product formation by hydrolysis of pyrophosphate. All amino acids were purchased from Sigma. Amino acids were added in a moderate amount to prevent aminoacylation with possible Ile contaminations in the non-cognate amino acid samples. Reactions were quenched after 30 min at 37°C by mixing with an equal amount of phenol/chloroform. AA-tRNA^Iles were purified by phenol/chloroform extraction followed by two consecutive steps on Bio-Spin P30 columns (Bio-Rad) and dialyzed against 10 mM NaOAc pH 4.5. Before further use AA-tRNA^Iles were renaturated by as described in Purification and labelling of tRNAs.

PRO-seq was performed as described previously (Mahat et al., 2016 (link)). 4 × 10⁶ OSCs treated with siRNAs for 96 hr were used for nuclei isolation. Isolated nuclei were resuspended in storage buffer and stored at −80°C until further processing. Nuclear run-on reactions were performed with biotin-11-CTP and biotin-11-UTP and unlabelled ATP and GTP. Purified RNA samples were fragmented for 10 min on ice in 0.2 N NaOH and purified using Bio-Spin P30 columns (Bio-Rad). Biotin-labelled RNA was purified using MyOne Streptavidin C1 Dynabeads (Thermo Fisher Scientific), decapped using the RppH enzyme (NEB), and purified by phenol/chloroform extraction. 3′ linkers and then 5′ linkers (same as for small RNA cloning) were ligated and biotinylated ligation products purified after each ligation using MyOne Streptavidin C1 Dynabeads. Reverse transcription was carried out using SuperScript III (Thermo Fisher Scientific), and libraries were amplified using HS Phusion Flex polymerase. The libraries were sequenced on an Illumina HiSeq 4000.

DNase I-digested total RNA (5 to 10 μg) was resuspended with the RNA loading buffer (95% formamide, 0.1% xylene cyanol, 0.1% bromophenol blue, 10 mM EDTA), denatured for 5 min at 95°C, were separated by electrophoresis on 10% polyacrylamide gel in denaturing condition (7 M urea) in 1X TBE buffer (10X TBE: 890 mM Tris base, 890 mM boric acid, 20 mM EDTA) for 5 h 15 at 300 V. To purify 16S and 23S degradation fragments, RNA from sucrose fractions were separated on a 1% agarose gel in 1X TBE buffer for 3 h 30 at 50 V. After staining with SYBR Safe stain (Invitrogen) and visualization on ChemiDoc imager (Bio-Rad), the bands corresponding to 5S, 5S*, and the different degradation fragments of 16S and 23S were cut and extracted from the gel in 0.3 ml RNA elution buffer (0.1 M sodium acetate (pH 6.5), 0.1% SDS, and 10 mM EDTA (pH 8)) and incubated with agitation ON at 6 to 10°C. After centrifugation at 14,000 rpm for 15 min at 4°C, RNA was purified using Bio-Spin P30 columns (Bio-Rad). The RNAs were precipitated with 1 volume of absolute ethanol, eluted with 30 μl of milliQ water (RNase-free), and then stored at −20°C.