Herring sperm dna

Cell cultures were washed twice with PBS and lysed in 500 µl ALP lysis buffer (10 mM glycine/0.1 mM MgCl₂/10 µM ZnCl₂/0.1 % Triton X-100) overnight at 4 °C. Of this lysate, 100 µl was added to 150 µl of LacZ assay buffer (0.1 M K₂HPO₄/0.1 % Triton X-100/0.02 M Na₂HPO₄/2.2 mM KCl/0.2 mM MgSO₄/7 mM 2-mercaptoethanol) containing 1 mg/ml o-nitro-phenyl-β-D-galactopyranoside (ONPG) and incubated for 3 h at 37 °C. The amount of o-nitro-phenol formed was determined by measuring the absorption of the assay solution at 405 nm using a ThermoMax microplate reader (Molecular Devices, Sunnyvale, CA, USA). The β-galactosidase activity was corrected for the amount of DNA in the cultures. To release the DNA from the cell debris, the remaining cell lysate was incubated with an equal volume of SCC buffer containing 100 µg/ml proteinase K (Invitrogen, Carlsbad, CA, USA) for 40 h at 56 °C. DNA content was measured using Hoechst 33258 (ICN Biomedicals, Inc., Irvine, CA, USA) and calibrated against a DNA standard (0.5–10 µg/ml herring sperm DNA; Invitrogen).

Hex-labeled probes were generated using the primer pairs listed in Table S3 and plasmids containing the WT or mutant promoters as the templates. Assays were performed as described (84 (link)). Briefly, purified His₆-MrpC was mixed at the indicated concentrations with 6 nM (dmxB fragments) or 10 nM (pmxA fragments) of Hex-labeled DNA fragment in reaction buffer (10 mM Tris [pH 8.0], 50 mM KCl, 1 mM dithiothreitol [DTT], 10 μg · mL⁻¹ bovine serum albumin [BSA], 10% glycerol, 0.5 μg herring sperm DNA [Thermo Fisher Scientific]) in a total volume of 10 μl and incubated for 15 min at 20°C. Reaction samples were separated on a 5% polyacrylamide gel in 0.5× Tris-borate-EDTA (TBE; 45 mM Tris, 45 mM borate, and 1 mM EDTA) for 1.5 h. Gels were imaged using a Typhoon phosphoimager (GE Healthcare).

RNA was isolated from BM cells using a Direct-zol RNA miniprep plus kit (Zymo Research) according to the manufacturer’s protocol. Subsequently, DNA was digested using a Turbo DNA-free kit (Thermo Fisher) following the manufacturer’s protocol. The RNA was recovered and concentrated using an RNA clean and concentrator-5 kit (Zymo Research). Northern blot analysis was performed according to standard procedures. Briefly, 5 µg of total RNA was run on a 1% denaturing agarose gel. The RNA was transferred to a Hybond-N+ membrane (GE Healthcare) by capillary transfer and fixed by ultraviolet-light crosslinking. The membrane was pre-hybridized at 42 °C for 20 min and then hybridized in PerfectHyb plus hybridization buffer (Sigma) containing 1 × 10⁶ cpm ml⁻¹ of ³²P-labelled TERRA DNA probe 5′-(TAACCC)5-3′ and 0.1 mg ml⁻¹ herring sperm DNA (Thermo Fisher). Hybridization was carried out overnight at 42 °C. The following day, the membrane was washed once in low stringency buffer (2×SSC and 0.1% SDS) at room temperature for 5 min and twice in high stringency buffer (0.5×SSC and 0.1% SDS) at 42 °C for 20 min. The membranes were exposed to muliautoradiography film at −80 °C. After exposure, the membrane was stripped in boiling stripping buffer (0.1% SDS and 5 mM EDTA) and re-hybridized using a GAPDH probe (5′-GTAGACCCACGACATACTCAGCACCGGCCTCACCCCATT-3′) as a loading control.