NextSeq 500 paired end 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).  Cells were treated with retapamulin (Sigma CDS023386) at a final concentration of 0.125 mg/ml for 15 minutes at room temperature, with occasional manual shaking. 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. Monosomes were run on a 15% denaturing gel alongside a 31 nt RNA oligo to size-select 31±5 nt fragments. Samples were gel-extracted in 500 ml RNA gel extraction buffer (300 mM NaOAc (pH 5.5), 1 mM EDTA, 0.1 U/mL SUPERase-In RNase inhibitor) followed by isopropanol precipitation. Samples were dephosphorylated by incubating with T4 PNK for 1 hour at 37 °C, before being phenol:chloroform:isoamyl alcohol extracted and ethanol precipitated. Samples were then ligated to the 3' linker oligo JW9371 using T4 RNA Ligase 2 (truncated, K227Q) at a 1:4 RNA:linker ratio. The ligation reaction was incubated for 3 hours at 37 °C, followed by 20 minutes at 65 °C. The reaction was run on a 15% denaturing gel alongside a control RNA oligonucleotide (JW9370) of the expected size of the ligated product. The sample ligation corresponding to the size of the control RNA was extracted in 500 mL RNA gel extraction buffer. Reverse transcription was performed on the RNA samples using Superscript III (Life Technologies) and oligo JW8875, as described previously (34). The reaction was run on a 10% denaturing gel and extracted in 500 μL DNA gel extraction buffer (300 mM NaCl, 10 mM Tris-Cl (pH 8), 1 mM EDTA). Reverse-transcribed cDNA was circularized using CircLigase, and PCR-amplified as described previously (34). Between 4 and 9 cycles of PCR were performed using Phusion High Fidelity DNA Polymerase, JW8835 as the standard forward primer and JW3249, 3250, 8876 or 8877, corresponding to Illumina index numbers 1, 2, 34 or 39 respectively, as the reverse primer. 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. Following an isopropanol precipitation, samples were quantified and sent for sequencing.
Experimental Factor: compound
retapamulin
Experimental Factor: dose
0.125