Illumina HiSeq 2500 sequencing; Pervasive, non-functional translation in mycobacterium tuberculosis
Pervasive, non-functional translation in mycobacterium tuberculosis
Cells were collected by filtration over a 0.22 μm filter. Cells were flash frozen in liquid nitrogen with 0.7 ml lysis buffer (20 mM Tris pH 8.0, 10 mM MgCl2, 100 mM NH4Cl, 5 mM CaCl2, 0.4 % Triton X100, 0.1 % NP-40, 1 mM chloramphenicol, 100 U/mL DNase I). Frozen cells were milled using a Retsch MM400 mixer mill for 8 cycles of 3 minutes each at 15 Hz. Milling cups were re-frozen in liquid nitrogen in between each milling cycle.  M. tuberculosis MC27000 cells were grown in 7H9 medium supplemented with 10% OADC (Oleic Albumin Dextrose Catalase), 0.2 % glycerol, 100 ug/ml pantothenic acid and 0.05 % Tween80 at 37 °C, without shaking. An initial culture was set up in 10 mL and grown until OD600 = approx. 0.4. This culture was added to 400 mL media and grown until OD600 = 1.0 (2-3 weeks).  Cell extracts were thawed and incubated on ice for 30 min. Samples were spun, supernatants removed and passed over a 0.22 μm filter then quantified by nanodrop and 1 mg aliquots of cell extracts were flash-frozen in liquid nitrogen. Monosomes were isolated by digesting 1 mg of cell extract with 1,500 units of micrococcal nuclease for 1 hour at room temperature on a rotisserie rotator. The reaction was quenched by adding 2 μL 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. The RNA molecules were dephosphorylated by treatment with T4 polynucleotide kinase (New England Biolabs). Then polyacrylamide gel purification was performed for size selection of ~28 nt RNA fragments. Approximately 25–30 nt poly-A tails were added to recovered RNA fragments with an Ambion poly(A) tailing kit (Life Technologies) following the manufacturer's protocol. The polyadenylated RNA samples were reverse transcribed using SuperScript III (Life Technologies), and primer JW2364 (/5Phos/GATCGTCGGACTGTAGAACTCTGAACCTGTCGGTGGTCGCCGTATCATT/iSp18/CACTCA/iSp18/CAAGCAGAAGACGGCATACGATTTTTTTTTTTTTTTTTTTTVN)[57]. The reverse transcription products were circularized by CircLigase ssDNA ligase (Epicentre). For the footprint libraries, ribosomal RNAs were subtracted from circularized ssDNA using biotinylated sense-strand oligonucleotides JW4267 (/5Biosg/TATCCTGAGAGGTGATGCATAGCCG), JW4268 (/5Biosg/TAAACGGTGGGTACTAGGTGTGGGTTTC), JW4269 (/5Biosg/CTTGGGATCCGTGCCGTAGCTAACGCAT), JW4270 (/5Biosg/AGGAAGGTAGCCGTACCGGTCAGTG), and JW4271 (/5Biosg/CACACCGCCGAAGCCGCGGCAGCCAAC). PCR amplification was performed using the circularized cDNA as template, JW2365 AATGATACGGCGACCACCGA) and JW2366 (CAAGCAGAAGACGGCATACGA) as primers[57], and Phusion High-Fidelity DNA Polymerase (New England Biolabs). The PCR amplified DNA libraries were deep sequenced.
Experimental Factor: compound
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