title: GSM3258509: Staphylococcus aureus, DM, replicate 2; Staphylococcus aureus; ncRNA-Seq
library_construction_protocol: We lysed pelleted bacteria in 400μL of 0.3M NaOAc/HOAc, 10mM EDTA pH 4.8 with an equal volume acetate saturated phenol chloroform pH 4.8. We placed samples in a reciprocating bead beater for 45 seconds on  maximum intensity using FastPrep lysing matrix B. We centrifuged samples at 21,000rcf for 15  minutes at 4°C before re-extraction and ethanol precipitation of total RNA.Total tRNA was subsequently isolated using a denaturing 10% polyacrylamide gel followed by passive gel elution and ethanol precipitation. Homemade protocol based on Illumina protocols. Deacylated RNAs with or without the demethylation were first treated with T4 Polynucleotide Kinase (Epicentre) at 37C° for 30 min to further warrant a free 3' hydroxyl group for template switching. Template-switching reactions were performed as described7. Briefly, we used an initial template-primer substrate consisting of a 41-nt RNA oligonucleotide (5'-AGA UCG GAA GAG CAC ACG UCU AGU UCU ACA GUC CGA CGA UC/3SpC3/-3') that contains Illumina Read1 and Read2 primer-binding sites and a 3' blocking group (three carbon spacer; IDT) annealed to a complementary 32P-labeled DNA primer with a single-nucleotide 3' overhang, T, which facilitates the template switch to full-length tRNA that mostly contain a 3' CCA end. For g2RT template-switching reactions, average 100 ng of demethylated tRNAs or 1 μg of demethylated total RNA were mixed with the initial template-primer substrate (100 nM) and g2RT (1 μM of TeI4cΔEn or 500 nM of GsI-IIC RT) in reaction medium containing 450 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl, pH 7.5, and DTT (1 mM for TeI4cΔEn or 5 mM for GsI-IIC RT). For template-switching with TeI4cΔEn RT, the reactions were pre-incubated at room temperature for 30 min, initiated by adding 25 mM dNTPs to a final concentration of 1 mM and incubating at 60°C for 30 min. For template-switching with GsI-IIC RT, the pre-incubation step is not required. The reactions were terminated by adding 5 M NaOH to a final concentration of 0.25 M, incubating at 95°C for 3 min, and neutralizing with 5M HCl. The cDNAs resulting from template switching were analyzed in a denaturing 6% polyacrylamide gel, electroeluted using a D-tube Dialyzer Maxi with MWCO of 6-8 kDa (EMD Millipore), and ethanol precipitated with 0.3 M sodium acetate in the presence of 25 µg of linear acrylamide (Life Technologies). The purified cDNAs were then circularized with CircLigase II (Epicentre) using manufacture's protocol with extended incubation time of 5 hours at 60°C, phenol-CIA extracted, ethanol precipitated and amplified with Phusion-HF (Thermo Scientific) using Illumina multiplex (5'- AAT GAT ACG GCG ACC ACC GAG ATC TAC ACG TTC AGA GTT CTA CAG TCC GAC GAT C -3') and barcode (5'- CAA GCA GAA GAC GGC ATA CGA GAT BARCODE GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T -3') primers for 12 cycles of 98°C for 5 sec, 60°C for 10 sec and 72°C for 10 sec. The PCR products were sequenced on Illumina HiSeq 2000 system.
attributes.tag: GEO Accession
attributes.value: GSM3258509