Dig wash and block buffer set

We performed Northern blotting using NorthernMax Kit (Thermo Fisher Scientific, California, USA). Briefly, RNA (15 μg for detection of endogenous circCUL2 and 8 μg for detection of linear CUL2) was denatured with 3 volumes formaldehyde load dye (Ambion) for 15 min at 65°C and loaded on 1% agarose gel. After the electrophoresis, RNA was transferred on Hybond N+membrane (GE Healthcare, Uppsala, Sweden) by capillary transfer. Transferred RNA was ultraviolet-crosslinked (at 265 nm). Pre-hybridization was performed at 68°C for 30 min and hybridization was performed at 68°C overnight. The membrane was washed with 2 × SSC 0.1% SDS twice 5 min at room temperature, then twice 15 min with 0.1 × SSC 0.1% SDS at 68°C. The membrane was hybridization with anti-DIG antibody and washed using DIG Wash and Block Buffer Set (Roche, Indianapolis, IN, USA). After washing, the blot was detected with the DIGluminescence detection kit (Roche). DIG-labeled probes were prepared using DIG Northern starter Kit (Roche) by in vitro transcription with PCR products as templates for T7 transcription.

The authors performed northern blotting using NorthernMax Kit (Thermo Fisher Scientific, Carlsbad, California, USA). Briefly, RNA (15 µg for detection of endogenous circARHGAP35 and 8 µg for detection of linear ARHGAP35) was denatured with 3 volumes formaldehyde load dye (Ambion) for 15 min at 65 °C and loaded on 1% agarose gel. After the electrophoresis, RNA was transferred on Hybond N+ membrane (GE Healthcare, Uppsala, Sweden) by capillary transfer. Transferred RNA was ultraviolet‐crosslinked (at 265 nm) at 200000 µJ cm⁻². Pre‐hybridization was performed at 68 °C for 30 min and hybridization was performed at 68 °C overnight. The membrane was washed with 2× SSC 0.1% SDS twice 5 min at room temperature, then twice 15 min with 0.1× SSC 0.1% SDS at 68°C. The membrane was hybridization with anti‐DIG antibody and washed using DIG Wash and Block Buffer Set (Roche, Indianapolis, IN, USA). After washing, the blot was detected with the DIG luminescence detection kit (Roche). DIG‐labeled probes were prepared using DIG Northern starter Kit (Roche) by in vitro transcription with PCR products as templates for T7 transcription.

60 μg of RNA per lane in Gel loading buffer II (Ambion) were denatured at 95°C for 5 min prior to loading. RNA was size separated on a 15% PAA, 8 M Urea TBE gel and transferred onto a positively charged nylon membrane (Roche) at 50 V for 50 min in TBE buffer at 4°C. After UV cross-linking (120 mJ / cm²) DIG-labelled DNA probes (S1 Table, labelled using the 2^nd Generation DIG-Tailing Kit [Roche]) were hybridized over night at 42°C in DIG EasyHyb Buffer (Roche). For development the DIG Wash and Block Buffer Set (Roche) and CDP-STAR (Roche) were used according to the manufacturer’s instructions. CDP-STAR signal was recorded using the Intas Chemostar Imager system.

GFP selection was used to identify transgenic plants. To confirm gene insertion, DNA was isolated from GFP-positive plants and checked using PCR. DNA-positive lines were then subjected to Southern blot using the digoxigenin (DIG) wash and block buffer set from Roche (IN, USA). The PCR DIG probe synthesis kit from Roche (IN, USA) was used to generate the DNA probe against the Cauliflower mosaic virus (CaMV) 35S promoter (Hart and Basu, 2009 (link)). Around 20 µg of genomic DNA was digested at 37 °C with NdeI for 18 h. Digested product was electrophoresed on a 1% (w/v) agarose gel at 40 V for 5 h. Next, the gel was transported to a nylon membrane and hybridized with the CaMV 35S promoter probe using a DIG DNA labelling and detection kit (Roche). This was followed by antibody binding and chemiluminescent reaction change by CDP star. Lastly, a film was used for exposing the membrane for analysis of the number of T-DNA insertions visualized by the ChemiDoc™ Touch Imaging System (Bio-Rad).

For each sample, total RNA from 5 brains, from which retinae were removed, of each stage from L5F to adult stage were extracted using TRIZOL Reagent (Invitrogen). Total RNA were also extracted from female embryos, male embryos, queen’s ovary or abdomen. These sample RNA were stored at -80 °C until use. The DIG-labeled sense or antisense riboprobes of Nb-1 RNA were prepared as described above.
Northern blotting analysis was performed according to a standard protocol using DIG-labeled riboprobes with slight modification. Sample RNA were subjected to denaturing urea-polyacrylamide gel (5.0% polyacrylamide, 8 M urea) electrophoresis, and transferred to a nylon membrane. The membranes were hybridized with the riboprobes of Nb-1 RNA at 68 °C in the hybridization mixture consisting of 50% formamide, 10 × Denhardt’s solution (Wako), 20 mM sodium phosphate (pH 6.5), 750 mM sodium chloride, 75 mM sodium citrate, 0.5% sodium dodecyl sulfate, and 100 μg/ml Herring sperm DNA (Promega). After washing, chemiluminescent detection was performed using DIG Wash and Block Buffer Set (Roche), antibody against DIG conjugated to alkaline phosphatase (Roche), and CDP-Star (Roche). Band intensities were quantitatively analyzed by ImageJ software version 1.54f. (https://imagej.net/ij, National Institutes of Health, Bethesda, MD).

A total of 1 μg of U2OS RNA and 5 μg of HME or HELA RNA were denatured in 50% formamide by heating at 65 °C for 15 min, incubated for 2 min on ice, and spotted onto a positively charged nylon membrane. Membranes were UV-crosslinked (120 mJ) and hybridized overnight in ULTRAhyb hybridization buffer (Invitrogen) with DIG-labeled TERRA (CCCTAA)₅ probe at 42 °C. Membranes were washed and blocked using a DIG wash and block buffer set (Roche). The hybridization signal was revealed using anti-DIG-alkaline phosphatase antibody (Roche) and CDP-Star (Roche). DIG-labeled 18 S rRNA probe (5′-CCATCCAATCGGTAGTAGCG-3′) was used for normalization.
DNA samples (ChIP/DRIP experiments) were denatured at 95 °C for 10 min, incubated on ice for 2 mins, and slot blotted onto nylon membranes. Hybridization and detection steps were performed as described above. Telomere DNA was detected with either DIG-(TTAGGG)₅ probe (for the C-rich strand) or (CCCTAA)₅ probe (for the G-rich strand). An Alu probe (5′-GTGATCCGCCCGCCTCGGCCTCCCAAAGTG-3′) was used for ChIP experiments.