GSM7688105: Mtb_truB_CMC; Mycobacterium tuberculosis; ncRNA-Seq
Extraction of total RNA: Strains were grown to mid-log phase with the appropriate antibiotics and inducing agents described above. RNA was collected at the same OD600 for each strain (between 0.4-0.6). Cells were left on ice for 20 minutes, then pelleted by centrifuging at 4,000 rpm for 10 minutes at 4 °C. Pellets were resuspended in 0.5-1 mL of TriZol (Life Technologies) and lysed using a BeadBug microtube homogenizer (Millipore Sigma). 200 uL of chloroform was added to each tube, after which samples obtained from Mtb strains were removed from biosafety level 3 precautions. Samples were centrifuged at 15,000 rpm for 15 minutes at 4 °C and the aqueous layer was collected into a fresh tube. To the original tube, 250 uL of sodium acetate buffer (300 mM sodium acetate pH 5.2 and 10 mM EDTA pH 8.0) was added, and samples were vortexed at 4 °C for 5 minutes then centrifuged at 15,000 g for 15 minutes at 4 °C. The aqueous layer was added to the fresh sample-containing tubes. 400 uL chloroform was added, and tubes were briefly vortexed and then centrifuged at 15,000 rpm for 1 minute at 4 °C. The aqueous phase was collected into a fresh tube and RNA recovered by ethanol precipitation. RNA pellets were resuspended in 10 mM sodium acetate pH 5.2 and stored at -80 °C until processed for sequencing. Total RNA samples were alkali-treated prior to tRNA extraction to deacylate all tRNAs (1 hour at 37 °C in 100 mM Tris-HCl pH 9.0).  Isolation of tRNA fraction: 1-2 ug of total RNA was run on a 10% TBE-UREA gel (ThermoFisher Scientific) at 250 V for 1 hour. Gels were stained with SYBR Gold (ThermoFisher Scientific), and tRNA was excised. Excised gels containing tRNA fractions were mashed in RNAse-free tubes, and 300 uL elution buffer (300 mM NaOAc pH 5.5, 1 mM EDTA pH 8.0, 0.10% SDS) was added to each tube. Samples were shaken on a thermoshaker (Eppendorf) for 1-4 hours at 37 °C and supernatant was collected using an Ultrafree filter column (Millipore Sigma). tRNA was recovered by isopropanol precipitation. tRNA dephosphorylation: tRNA was dephosphorylated using QuickCIP (New England BioLabs) according to manufacturer instructions, and tRNA was collected by phenol-chloroform extraction followed by isopropanol precipitation.   Iodoacetamide (IAA) treatment: IAA treatment was performed as described. Briefly, 500 ng of total RNA is combined with 10 mM of iodoacetamide, 50 mM NaPO4 pH 8.0, and 50% DMSO in a final volume of 50 uL. Reactions were incubated at 50 °C for 15 minutes and quenched with DTT.  CMC treatment: CMC treatment was carried out as described in ref. Briefly, 2.5 ug tRNA fraction in 0.5 ul was mixed with 15 ul CMC-BEU buffer with or without CMC (0.34 M or 0 M CMC, 7 M urea, 4 mM EDTA, and 50 mM bicine pH 7.9) and incubated at 37 °C for 20 min. Adding 100 ul CMC stop solution (0.3 M NaOAc pH 5.2 and 100 mM EDTA) quenched the reaction. RNA was desalted with PD-10 desalting column (Cytiva) and recovered by ethanol precipitation. RNA was dissolved in 40 ul of 50 mM sodium carbonate buffer (pH 10.4) and incubated at 37 °C for 4 hours, followed by ethanol precipitation.  Adapter ligation: 0.5 uL RNase inhibitor was added to 3.5uL dephosphorylated tRNA (200-250 ng tRNA) and samples were boiled at 80 °C for 2 minutes. Boiled tRNA was mixed with 12 uL PEG buffer mix (10 uL 50% PEG8000, 2 uL 10 x buffer B0216S; New England Biolabs). 3 uL of 5' adenylated linkers (Supplementary Table 4) were added (33 pmol/uL) along with 1 uL T4 RNA ligase 2 truncated (New England BioLabs) and incubated at 25 uC for 2.5 hours. Samples were recovered by isopropanol precipitation and run on a 10% TBE-Urea PAGE gel for 40 minutes at 250 V. Ligated products were recovered by gel excision as described above.  Reverse transcription: Identical quantities of samples with different adapter sequences were pooled for reverse transcription for a total of 200250 ng tRNA. Reverse transcription was performed by combining 2.1 uL dephosphorylated tRNA with 100 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 1.25 uM RT primer, 450 mM NaCl, 5 mM MgCl2, 5 mM DTT, 500 nM TGIRT (InGex), and 15% PEG8000 in a final volume of 9 uL. Samples were incubated at 25 °C for 30 minutes, after which 1 uL 10 mM dNTPs (New England BioLabs) were added and reactions incubated at 60 °C for 1 hour. 1.15 uL NaOH was added, and samples were boiled for 15 minutes and run on a 10% TBE Urea PAGE gel at 250V for 1 hour. Reverse transcription products were excised from gels and cDNA recovered by isopropanol precipitation. Linear single-stranded cDNA was circularized using CircLigase II (Lucigen) in accordance with manufacturer instructions.  PCR of tRNA libraries: PCR reactions were set up using HF Phusion according to the manufacturer's instructions using a universal reverse primer and a different index primer for each pool of samples. PCR reactions were aliquoted into 4 tubes and collected after 6, 8, 10, and 12 cycles. Samples were run on a Native TBE PAGE gel (ThermoFisher Scientific) at 180 V for 50 minutes, and amplified products were cut from the same cycle for each sequencing run. Samples were recovered by gel excision and isopropanol precipitation.