Mycolicibacterium smegmatis MC2 155
Protocols: 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. RNA from a scoopful of cell powder was extracted with acid phenol:chloroform followed by isopropanol precipitation as described previously. DNase treat total RNA: set up 50uL reaction including 6-8 ug RNA, 5 uL of 10X buffer and 1 uL DNase enzyme (Ambion). Incubate 45 min at 37 °C. Stop reaction by adding 352 uL DEPC H2O + 134 uL acid phenol:chloroform + 268 uL chloroform:isoamyl alcohol 24:1. Transfer all to phase lock tube and spin at max speed for 6 min. Transfer upper phase to new microcentrifuge tube containing 40 uL 3M Na Acetate + 2 uL glycogen (20 mg/mL stock). Add 1 mL ice cold EtOH and precipitate at -80 °C overnight. Spin samples for 30 min at 4 °C at max speed. Pipet off supernatant. Wash with 0.7 mL of 70% EtOH (in DEPC H2O), spin 10 min at max speed at 4 °C and remove supernatant. Wash again with 0.7 mL of 70% EtOH (in DEPC H2O), spin for 10 min at max speed at 4 °C and pipet off supernatant. Dry pellet at room temperature and resuspend in 15 uL nuclease free water. Remove ribosomal RNA using the NEBNext rRNA Depletion Kit (NEB # E7850): input is 1 ug DNase treated RNA. For mycobacteria, adjust 15 uL probe hybridization reaction to 1 ug RNA in 10 uL + 1 uL NEBNext Bacterial rRNA Depletion Solution + 2 uL NEBNext Probe Hybridization Buffer + 2 uL mycoprobe. Incubate (in thermocycler) 95 °C 2 min, ramp down to 22 °C @ 0.1 °C/sec, 22 °C 5 min. Set up RNase H digestion with 15 uL hybridized RNA: add 2 uL RNase H Reaction buffer, 2 uL NEBNext thermostable RNase H and 1 uL nuclease free water. Incubate in thermocycler 30 min @ 50 °C with heated lid set to 55 °C. Set up DNase I digestion with 20 uL RNA: add 5 uL DNase I reaction buffer, 2.5 uL NEBNext DNase I and 22.5 uL nuclease free water. Incubate in thermocycler 30 min @ 37 °C with heated lid set to 40 °C. Clean reaction using NEBNext RNA Sample Purification beads: add 90 uL beads to RNA and mix by pipetting 10x. Incubate 15 min on ice. Place tube on magnetic stand and when solution clear, pipet off supernatant. Add 200 uL 80% EtOH to tube (do not disturb pellet), incubate 30 sec @ RT, then remove supernatant. Repeat for a second wash. Pipet off residual EtOH and air dry beads up to 5 min with tube on rack. Remove tube from rack and resuspend beads in 25 uL nuclease-free water. Pipet 10x to mix, incubate 2 min @ RT, place tube on magnetic stand until solution clears, transfer supernatant to fresh tube. 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 25 minutes at 95°C followed by isopropanol precipitation. rRNA depleted (+/- fragmentation) mRNA was 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.
ENA-FIRST-PUBLIC
2023-04-07
organism
Mycolicibacterium smegmatis MC2 155
organism
Mycolicibacterium smegmatis MC2 155
ENA-LAST-UPDATE
2023-04-07
growth condition
7H9 medium
scientific_name
Mycolicibacterium smegmatis MC2 155
organism part
whole organism
developmental stage
exponential phase
genotype
Δmsmeg_0945
ENA-FIRST-PUBLIC
2023-04-07
ENA-LAST-UPDATE
2023-04-07