NextSeq 500 paired end sequencing; Ribosome profiling performed in Mycobacterium smegmatis MC2 155wild type and ORF MSMEG_0945 MSMEG_1916 mutants
Ribosome profiling performed in Mycobacterium smegmatis MC2 155wild type and ORF MSMEG_0945 MSMEG_1916 mutants
Cells were collected by filtration over a 0.22 um filter.  Cells were scraped off the filter using a cell scraper and flash frozen in liquid nitrogen + 700 ul lysis buffer (20mM Tris pH 8.0; 10mM MgCl2; 100mM NH4Cl; 5mM CaCl2; 0.4% Triton X100; 0.1% NP-40; 1mM chloramphenicol; 100 U/ml DNase I).  Frozen cells were milled for 6 cycles (MG1655, mc2-155 and MKD158) or 8 cycles (mc2-7000) of 3 minutes each at 15 Hz.  Pulverized cells were transferred to a 50 mL conical containing liquid nitrogen and stored at -80°C. Overnight cultures were set up in 4 mL 7H9 media with 0.2% glycerol, 10% OADC and 0.05% Tween80 and grown at 37°C/225 rpm.  Subcultures were set up in 400 mL of the same media at OD600 ~0.01 and grown to OD600 ~ 1.0. Cell extracts were thawed and incubated on ice for 30 min.  Samples were transferred to a new 1.7 mL tube and spun at 13,200 rpm for 10 min at 4°C.  Supernatants removed and transferred to new tube.  For all samples, 1 uL of supernatant was diluted 1:100 in DEPC H2O and quantified by nanodrop.  1 mg aliquots of cell extracts were flash frozen in liquid nitrogen.  Monosomes were isolated by digesting 1 mg of cell extract with 1500 units of MNase for 1 hour at room temperature on a rotisserie.  The reaction was quenched by adding 2 ul of 0.5 M EGTA after which the digest was run over a 10-50% sucrose gradient.  Fractions from the sucrose gradients were run on a 1% agarose gel with 1% bleach.  Based on the gel, fractions were selected and monosomes isolated by acid phenol:chloroform extraction and isopropanol precipitation. Monosomes were denatured at 80°C, 1400 rpm for 2 min and then run on a 15% denaturing gel alongside a 15nt RNA oligo and a low range ssRNA ladder to size select approximately 15 nt-50 nt fragments.  Samples were gel extracted overnight at 4°C on a nutator in 500 ul RNA gel extraction buffer (300mM NaOAc (pH 5.5), 1 mM EDTA, 0.1 U/ml SUPERase-In) followed by isopropanol precipitation.  Samples were denatured at 80°C for 90 sec and then equilibrated to 37°C.  Samples were dephosphorylated by incubating with T4 PNK for 1 hour at 37°C and isopropanol precipitated.  Approx 1 ug of the preadenylated 3' linker oligo JW9371 was added to each sample and samples were denatured at 80°C for 90 sec.  RNA and JW9371 linker oligo were added to a ligation reaction using T4 RNA Ligase 2, truncated KQ in the presence of 15% w/v PEG8000.  The ligation reaction was incubated o/n at 16°C, followed isopropanol precipitation.  Samples were denatured at 80°C, 1400 rpm for 2 min and then run on a 15% denaturing gel alongside a mock ligation and a 31 nt marker, and the sample ligation from 31 nt-80 nt was extracted overnight at 4°C on a nutator in 500 uL RNA gel extraction buffer.  Reverse transcription was performed on the RNA samples using Superscript III (Life Technologies) and oligo JW8875 at 55°C for 30 min according to the protocol of Ingolia.  Samples were hydrolyzed by adding 1N NaOH ([final] = 0.1N) and incubating at 95°C for 15 min followed by 80°C for 2 min.  The reaction was run on a 10% denaturing gel and was extracted overnight at 4°C on a nutator in 500 ul DNA gel extraction buffer (300 mM NaCl, 10 mM Tris-Cl (pH 8), 1 mM EDTA).  Reverse transcribed samples were then circularized using CircLigase and incubated for 1 hour 30 min at 60°C followed by a 10 minute incubation at 80°C.  Samples were PCR amplified according to Ingolia using Phusion High Fidelity DNA Polymerase, JW8835 as the standard forward primer and JW3249, 3250, 8876, 8877, 10754, 10755, 10756 or 10757 corresponding to barcode index numbers 1, 2, 34, 39, 40, 41, 42 or 43 respectively, as the reverse primer.  A range of cycle numbers of PCR were tested.  Samples were run on an 8% acrylamide gel and were selected for gel extraction based on the appearance of a distinct band above the adapter band on the gel.  Gel extraction was in 500 uL DNA gel extraction buffer, overnight at 4°C on a nutator.  Following an isopropanol precipitation, samples were quantified by Qubit and sent for sequencing.