Zeta probe gt membrane

Northern blots were performed using total RNA exactly as described previously (Melamed et al., 2020 (link)). For sRNAs, 5 μg of RNA were fractionated on 8% polyacrylamide urea gels containing 6 M urea (1:4 mix of Ureagel Complete to Ureagel-8 (National Diagnostics) with 0.08% ammonium persulfate) and transferred to a Zeta-Probe GT membrane (Bio-Rad). For longer RNAs, 10 μg of RNA were fractionated on a 2% NuSieve 3:1 agarose (Lonza), 1X MOPS, 2% formaldehyde gel and transferred to a Zeta-Probe GT membrane (Bio-Rad) via capillary action overnight. For both types of blots, the RNA was crosslinked to the membranes by UV irradiation. RiboRuler High Range and Low Range RNA ladders (Thermo Fisher Scientific) were marked by UV-shadowing. Membranes were blocked in ULTRAhyb-Oligo Hybridization Buffer (Ambion) and hybridized with 5′ ³²P-end labeled oligonucleotides probes (listed in Supplementary Table 8). After an overnight incubation, the membranes were rinsed with 2X SSC/0.1% SDS and 0.2X SSC/0.1% SDS prior to exposure on film. Blots were stripped by two 7-min incubations in boiling 0.2% SDS followed by two 7-min incubations in boiling water.

For smaller RNAs, total RNA (5 μg) was separated on a denaturing 8% polyacrylamide urea gel containing 6 M urea (1:4 mix of Ureagel Complete to Ureagel-8 (National Diagnostics) with 0.08% ammonium persulfate) in 1 X TBE buffer at 300 V for 90 min. The RNA was transferred to a Zeta-Probe GT membrane (Bio-Rad) at 20 V for 16 hr in 0.5 X TBE. For longer RNAs, total RNA (10 μg) was fractionated on formaldehyde-MOPS agarose gels as previously described (Adams et al., 2017 (link)). Briefly, RNA was denatured in 3.7% formaldehyde (Fisher), 1 X MOPS (20 mM MOPS, 5 mM NaOAc, 1 mM EDTA, pH 7.0) and 1 X RNA loading dye (Thermo Fisher Scientific) for 10 min at 70 °C and incubated on ice. The RNA was loaded onto a 2% NuSieve 3:1 agarose (Lonza), 1 X MOPS, 2% formaldehyde gel and separated at 125–150 V at 4 °C for 1–2 hr and then transferred to a Zeta-Probe GT membrane (Bio-Rad) via capillary action overnight (Streit et al., 2009 (link)). For both types of blots, the RNA was crosslinked to the membranes by UV irradiation. RiboRuler High Range and Low Range RNA ladders (Thermo Fisher Scientific) were marked by UV-shadowing. Oligonucleotide probes (listed in Supplementary file 3) for the different RNAs were labelled with 0.3 mCi of [γ-³²P] ATP (Perkin Elmer) by incubating with 10 U of T4 polynucleotide kinase (New England Biolabs) at 37 °C for 1 hr.

Northern blots were performed using total RNA exactly as described previously (Melamed et al., 2020 (link)). For small RNAs, 5 μg of RNA were fractionated on 8% polyacrylamide urea gels containing 6 M urea (1:4 mix of Ureagel Complete to Ureagel-8 (National Diagnostics) with 0.08% ammonium persulfate) and transferred to a Zeta-Probe GT membrane (Bio-Rad). For longer RNAs, 10 μg of RNA were fractionated on a 2% NuSieve 3:1 agarose (Lonza), 1X MOPS, 2% formaldehyde gel and transferred to a Zeta-Probe GT membrane (Bio-Rad) via capillary action overnight. For both types of blots, the RNA was crosslinked to the membranes by UV irradiation. RiboRuler High Range and Low Range RNA ladders (Thermo Fisher Scientific) were marked by UV-shadowing. Membranes were blocked in ULTRAhyb-Oligo Hybridization Buffer (Ambion) and hybridized with 5´ ³²P-end labeled oligonucleotides probes (listed in Supplementary file 4). After an overnight incubation, the membranes were rinsed with 2X SSC/0.1% SDS and 0.2X SSC/0.1% SDS prior to exposure on film. Blots were stripped by two 7 min incubations in boiling 0.2% SDS followed by two 7 min incubations in boiling water.

RNA samples (10–20 μg) were denatured for 10 min at 70°C in 98% formamide loading buffer, separated on 8 M urea–6% polyacrylamide gels, and transferred to Zeta‐Probe GT membranes (Bio‐Rad Laboratories) by electroblotting. To detect OxyS RNA, the membranes were hybridized with [³²P]‐end‐labeled oxyS primer (492) in modified CHURCH buffer (Church & Gilbert, 1984). To detect secE‐nusG full‐length RNA, samples (15 μg) were denatured for 10 min at 70°C in MOPS loading buffer, separated on 1.4% agarose gels, and transferred to Zeta‐Probe GT membranes by capillary transfer. secE‐nusG mRNA levels were detected using anti‐nusG‐labeled riboprobe synthesized using PCR template (2620 and 2554) as previously described (Hershko‐Shalev et al, 2016). Riboprobe hybridization buffer contained 50% formamide, 3.5% SDS, 250 mM NaCl, 82 mM Na₂HPO₄, and 40 mM NaH₂PO₄ at pH 7.2. After 2 h at 50°C, the membranes were washed for 20 min at 50°C in 2× SSC, 1% SDS, then for 20 min at 55°C in 1× SSC, 0.5% SDS, and last for 20 min at 60°C in 0.5× SSC, 0.1% SDS. tmRNA (10Sa) was used as a loading control (primer 1912).

Total RNA was extracted using TRIzol (Invitrogen) and digested with DNase 1 (Qiagen). 10 μg RNA was incubated with formamide (Sigma-Aldrich) at 65 °C for 10 min and separated in 12% acrylamide denaturing (urea) gels. The gels were stained with SYBR-Safe (Invitrogen) to visualize the RNA samples. RNA was transferred to Zeta-Probe GT membranes (Bio-Rad) and cross-linked by ultraviolet irradiation (Stratagene). The membranes were blocked in hybridization buffer (0.5 M Na₂PO₄ pH7.2, 15% formamide, 1% BSA and 7% SDS) for 30 min at 37 °C in a rotating hybridization oven. The miR-124 probes (5′-GGCATTCACCGCGTGCCTTA-3′) were labelled with γ-³²P ATP using PNK (NEB) and cleaned with a G25 column (GE Healthcare). Probes were heated at 65 °C before being added into the hybridization bottle, and membranes were hybridized at 37 °C overnight. The membranes were washed three times with 1 × SSC containing 0.1% SDS, and then was exposed to X-ray film for 1 day at −80 °C.

DNA probes were labeled with digoxigenin using the DIG-High Prime kit (Roche Applied Science). Membranes (Zeta-Probe GT membranes, Bio-Rad) were pre-hybridized in a 20 ml pre-hybridization solution (2× SSPE, 0.5% Blotto, 1% SDS, 10% dextran sulfate, and 0.5 mg/ml sonicated and denatured salmon sperm DNA) at 65°C for 4–6 h. Labeled DNA was added, and the hybridization lasted for 12–16 h. Hybridized membranes were sequentially washed with 2× SSC and 0.1% SDS at room temperature for 5 min twice and with 0.1× SSC and 0.1% SDS at 68°C for 15 min twice as well. Detection was performed with an antidigoxigenin-alkaline phosphatase-conjugated antibody (Roche Applied Science) and CDP-Star (PerkinElmer Life Sciences) according to the instructions provided by the manufacturers as described before [29 (link)]. All experiments were performed twice to confirm the results obtained were reproducible.