title: NextSeq 500 sequencing; Ribosome profiling of Mycobacterium tuberculosis under nutrient starvation
design: Ribosome profiling of Mycobacterium tuberculosis under nutrient starvation
library_construction_protocol: M. tuberculosis H37Rv was grown in Middlebrook 7H9 medium (BD Diagnostics) supplemented with 10% Middlebrook albumin-dextrose-catalase (ADC) (BD Diagnostics), 0.2% glycerol and 0.05 % Tween 80 at 37°C until OD600 was 0.4–0.6. For starvation experiments, exponentially growing bacteria were pelleted, washed twice in PBS and resuspended in PBS supplemented with 0.025% Tyloxapol, and maintained in culture for a further 24 h. Triplicate cultures of exponentially growing and nutrient starved cells were prepared for parallel RNA and Ribosome profiling experiments. For ribosome profiling experiments, chloramphenicol was added to a final concentration of 100 μg/ml two minutes prior to harvesting cells.  Sample preparation for ribosome profiling was carried out according to a modified form of the method of Latif et al.(Latif et al., 2015). All steps were performed on ice or using chilled buffers. M. tuberculosis cell pellets were resuspended in lysis buffer (20 mM HEPES, pH 7.4; 200 mM KCl, 1% TritonX-100, 12 mM MgCl2, 1 mM CaCl2, 1 mg/ml heparin, 100 μg/ml chloramphenicol and 5 U/ml DNase I)  and transferred to ribolysing matrix tubes (MPBio). Cell lysis was performed by ribolysing the sample three times for 30 s at power 6.5 (FastPrep-24, MPBio). Ribolysing matrix and cell debris were removed by centrifugation at 12,000 rpm (JA-15 1.5 rotor, Beckman) for 10 minutes.The ribosome profiling samples were then double filtered through a 0.2 m membrane (Corning) to ensure sterility before incubation for 45 minutes with 24 l microccocal nuclease (NEB) and 15 l DNaseI (Thermo Fisher) to digest mRNAs that are not bound by ribosomes . Nuclease digestion was stopped by addition of 10 l Superase In (Thermo Fisher) and 5 l 0.5 M EGTA and cell lysates loaded onto a 34% sucrose cushion. Ribosomes were harvested by centrifugation at 40,000 rpm for 15 h in an SW40Ti rotor (Beckman). To isolate the ribosome-bound mRNAs or ribosome footprints (RPF), the ribosomal pellet was then resuspended in Qiazol (Qiagen) and small RNA isolated using the miRNeasy mini kit (Qiagen). Isolated RNA was precipitated and resuspended in 10 mM Tris, pH 8.0. Finally, RPF samples were depleted of ribosomal RNA using the RiboZero rRNA removal kit for bacteria (Illumina) and purified using RNeasy MinElute columns (Qiagen). The quantity and quality of the RNA was assessed at each step using the bioanalyser and qubit.For RNA isolation, M. tuberculosis cell pellets were resuspended in RNApro solution (MPBio) and ribolysed three times for 30 s at power 6.5 (FastPrep-24, MPBio). Ribolysing matrix and cell debris were removed by centrifugation at 12,000 rpm (JA-15 1.5 rotor, Beckman) 4°C for 10 minutes. The total RNA sample was then removed from the ribolysing tube and chloroform added. The sample was vortexed for 10 s and incubated at room temperature for 5 minutes before centrifugation at 12,000 rpm (JA-15 1.5 rotor, Beckman) 4°C for 5 minutes. The aqueous phase was then transferred to a new tube and 1.5 volumes of 100% ethanol added. Samples were incubated at -20°C overnight and spun to pellet the RNA. The pellet was washed with 75% ethanol, air dried and resuspended in nuclease-free water. A further acid-phenol chloroform extraction was performed to improve purity of the RNA before resupending the final pellet in nuclease-free water. Genomic DNA contamination was assessed by PCR for 16S genomic DNA and, if present, samples were treated with DNase I followed by a further acid-phenol chloroform extraction.Next, samples were depleted of ribosomal RNA using the RiboZero rRNA removal kit for bacteria (Illumina) and purified using RNeasy MinElute columns (Qiagen). Finally, total RNA samples were then subjected to fragmentation using RNA fragmentation reagents (Thermo Fisher) at 80°C for 20 minutes. The quantity and quality of the samples were assessed at each step using the bioanalyser and qubit. RPF and total RNA samples were converted to cDNA libraries as previously described (Latif et al 2015). Briefly, samples were treated with T4 PNK (NEB) at 37ºC for 30 mins and purified using RNeasy MinElute columns (Qiagen). The NEBnext small RNA library prep kit (NEB) was used according to manufacturer's guidelines to construct the multiplexed cDNA libraries