Illustra microspin g 25 column

A PCR product corresponding to the promoter region of epsA was amplified using primers NSW52 (5′-TGG AGA ATT CTG TAC GGC TTG CAC TAA ATG TAC G-3′) and NSW53 (5′-GCC AGA ATT CGG ATC CAT TCA TAG CCT TCA GCC TTC CCG-3′), and a PCR product corresponding to the promoter region of tapA was amplified using primers NSW50 (5′-TGG CGA ATT CAT AGA CAA ATC ACA CAT TGT TTG ATC A-3′) and NSW51 (5′-GCC AGA ATT CGG ATC CAT CTT ACC TCC TGT AAA ACA CTG TAA-3′). The products were purified by gel extraction. As a positive control, the promoter region of aprE was amplified using primers NSW61 (5′-GGTAAAGCCTATGAATTCTCCATTTTCTTC-3′) and NSW654 (5′-GTCTAAGCTTGATCCACAATTTTTTGCT-3′). The promoter DNA was labeled using 50 μCi [γ-³²P]ATP (Perkin-Elmer) and T4 polynucleotide kinase (New England BioLabs). Unincorporated ATP was removed from the labeled DNA using an Illustra Microspin G-25 column (GE Healthcare). Phosphorylated purified DegU was produced as described previously (40 (link)), with the exception that 15 μM purified DegU∼P and 3.18 μM purified DegS (final concentrations) were added to the phosphorylation reaction mixture. The phosphorylation reaction, DNA binding, and mobility shift assay were performed as described previously (36 (link), 40 (link)).

Overall, 2–5 μg of total sRNAs were run on 18% denaturing polyacrylamide gel and transferred to a positively charged nylon membrane (GE Healthcare Amersham Hybond N+) on a Trans-Blot SD Semi-Dry Transfer Cell (Biorad). The RNA was ultraviolet-cross-linked to the membrane with Spectrolinker XL-1500 (Spectroline, ‘optimal crosslink'). Prehybridization was performed with Church Buffer (0.5 M NaH₂PO₄/Na₂HPO₄ pH 7.2, 1 mM EDTA, 7% SDS) at 37 °C overnight. 10 pmol of DNA probes (Supplementary Table 4) were labelled with T4 PNK (NEB) and 10 pmol [γ-³²P]-ATP (Hartmann Analytic) at 37 °C for 60 min. The labelled probes were purified with an Illustra MicroSpin G-25 column (GE Healthcare), mixed with 5 ml Church Buffer, and incubated with the membrane for 5 h at 37 °C. The membrane was rinsed once with 2x SSC buffer (0.3 M NaCl, 0.03 M sodium citrate, pH 7) and then washed three times with 2x SSC buffer for 15 min at 37 °C. The membrane was wrapped in cling film and exposed to a storage phosphor screen (BAS MS 2025—Fujifilm Corporation) overnight up to 2 days at −80 °C. The screen was scanned with a Typhoon FLA 9500 (GE Healthcare). For a second hybridization of the same membrane, the membrane was stripped in boiling 0.1% SDS for 5 min and subsequently prehybridized.

Nanoluciferase (Promega) was subcloned from pNL1.1 into pT7-5ʹ/3ʹMCS-A50 vector generated in house. pT7-NLuc-A50 was linearized with SacII and in vitro transcribed using T7 RiboMAX Express Large Scale RNA production System (Promega). Transcribed reporter RNAs were extracted with acid equilibrated phenol/chloroform and ethanol precipitated with 2.5 M ammonium acetate. Resulting RNAs were resuspended in DEPC-treated water and purified through Illustra MicroSpin G-25 column (GE Life Sciences). The resulting RNA was uncapped and contained a 50 nt synthetic poly(A) tail. In vitro translation assays were carried out in rabbit reticulocyte lysate system (Promega) following established protocols using 100 ng of in vitro transcribed RNA and 100 pmol of indicated tiRNA or control RNA. Reactions were incubated at 30 °C for 30 min. Luciferase activity was assayed using Nanoluc Luciferase assay Kit (Promega) on Luminometer (GloMax Explorer, Promega) according to manufacturer’s instructions (0.3 s measurement time).

The telomerase assay was as described^{12 (link)}. Reactions (20 μl) contained 1× human telomerase buffer, 1 μM oligonucleotide substrate, and dNTP mix as indicated in the figure legends. Reactions with cellular dNTP concentrations contained 24 μM dATP, 29 μM dCTP, 37 μM dTTP, 5.2 μM dGTP, and 0.3 μM 3000 Ci per mmol [α-³²P] dGTP or [α³²P] dTTP (PerkinElmer) as indicated. Reactions containing the modified dNTPs (Trilink Biotechnologies) substituted for their natural dNTP analog are indicated in the figure legends. The reactions were started by the addition of 3 μl (~35 fmol) of immunopurified telomerase eluent, incubated at 37 °C for 1 h, then terminated with 2 μl of 0.5 mM EDTA and heat inactivated at 65 °C for 20 min. ³²P-end labeled 18-mer loading control (8 fmol) was added to the terminated reactions before purification with an Illustra Microspin G-25 column (GE Healthcare). An equal volume of loading buffer (94% formamide, 0.1× TBE, 0.1% bromophenol blue, 0.1% xylene cyanol) was added to the reaction eluent from the G-25 spin column. The samples were heat denatured for 10 min at 100 °C and loaded onto a 14% denaturing polyacrylamide gel (7 M urea, 1× TBE) and electrophoresed for 90 min at constant 38 W. Samples were imaged using a Typhoon phosphorimager (GE Healthcare).