Illumina HiSeq 2500 sequencing; Pervasive 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. Total RNA from a small scoopful of cell powder was extracted with acid phenol:chloroform followed by isopropanol precipitation.  Ribosomal RNA was removed from 20 ug of total RNA using the Ribozero Kit (Illumina).   Fragmentation was performed by incubating the rRNA removed RNA with fragmentation buffer (0.5 M EDTA; 0.1 M Na2CO3; 0.1 M NaHCO3) for 30 minutes at 95°C followed by isopropanol precipitation. RNA was dephosphorylated using T4 PNK, 37°C for 1 hour followed by a phenol extracted/EtOH precipitation.  Samples were run on a 15% denaturing alongside JW512 as a 31 nt size marker and bands corresponding to 31 nt+/-10 nt were cut out and gel extracted overnight at 4°C in 500 uL RNA gel extraction buffer (300mM NaOAc (pH 5.5); 1mM EDTA; 0.1U/uL SUPERase-IN).  Approximately 25-30 nt poly-A tails were added to recovered RNA fragments using the 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). The reverse transcription products were circularized by CircLigase ssDNA ligase (Epicentre).  PCR amplification was performed using the circularized cDNA as template, JW2365 AATGATACGGCGACCACCGA) and JW2366 (CAAGCAGAAGACGGCATACGA) as primers, and Phusion High-Fidelity DNA Polymerase (New England Biolabs). The PCR amplified DNA libraries were deep sequenced.