Novex tbe urea gel

Northern blot was performed similarly as before^{72 (link),73 (link)}. Briefly, 1–5 μg purified total RNA or 5 pmole synthetic RNA oligos were resolved on 15% Novex TBE-Urea gel (Invitrogen #EC6885BOX) and transferred to Amersham Hybond-N + nylon membrane (Cytiva #RPN203B) by Trans-Blot SD semi-dry transfer apparatus (Bio-Rad). The membrane was cross-linked with 254 nm wavelength by Stratalinker (Strategene).
For Northern blot: After UV crosslinking, the membrane was blocked by ExpressHyb Hybridization Solution (Takara Bio #636833) and probed with biotinylated DNA probe (probe sequences see Supplementary Table 7) following the manufacturer’s instructions. Notably, the upper (U6 and full-length tRNA) and lower (tRF) parts of the membrane was cut after transfer, probed, and developed separately to avoid saturation of tRNA signals. Hybridized membrane was detected with Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher #89880).
For immuno-Northern blot: After UV crosslinking, the membrane was blocked with 3% milk in PBST and detected by m¹A primary antibody (MBL #D3453, used at 1:2000 dilution) followed by anti-mouse HRP-linked secondary antibody (Cell Signaling #7076, used at 1:5000 dilution). Chemiluminescence detection was performed with Immobilon HRP substrate (Millipore #WBKLS0500). Oligo size was compared to microRNA marker (NEB #N2102S) on the gel.

Increasing concentrations of both ring-nucleases from 10 nM to 150 nM were incubated with 25 nM of cOA₄ at 70 °C for 30 min followed by addition of 18 nM of SisCsx1 and 2.5 μΜ of RNA1 and further incubation at 70 °C for 5 min. The reactions were then separated using 15% Novex TBE-urea gel (Invitrogen).

Total RNA was extracted from adult testes (wild-type and Mvh homozygote) and organ cultured tissues (4-day culture) using Sepasol-RNAI SuperG (Nacalai Tesque) according to the manufacturer’s manual. A total of 20 μg of total RNA was electrophoresed on a 10% Novex^TM TBE-Urea Gel (Invitrogen) and transferred to a Hybond-N+ membrane (Amersham Biosciences) using semi-dry electroblotter at 400mA for 30 min and subsequently UV crosslinked. Oligo DNA probes (10 ng each) were labeled using gamma [³²P]-ATP with a MEGALABEL Kit (Takara-Shuzo). Hybridization was performed in a hybridization buffer (10% SDS, 10% dextran sulfate, 1M NaCl, 0.5mg/ml sonicated salmon sperm DNA) at 65℃ overnight. The membranes were then washed twice in 2xSSC and 0.5% SDS at room temperature for 30min and in 0.2xSSC and 0.5% SDS at 65℃ for 30 min. Next, the signals were visualized using a BAS-3000 image-analyzer (GE-HealthCare).

A total of 20 μg of total RNA was electrophoresed on a 10% NovexTM TBE-Urea Gel (Invitrogen) and transferred to a Hybond-N+ membrane using a semi-dry electroblotter at 400 mA for 30 min, followed by UV crosslinking. Oligo DNA probes (10 ng each) were labeled with gamma [32P]-ATP using a MEGALABEL Kit (Invitrogen). Hybridization was performed in hybridization buffer (10% SDS, 10% dextran sulfate, 1 M NaCl, 0.5 mg/mL sonicated salmon sperm DNA) at 65 °C overnight. The membranes were washed twice in 2x SSC and 0.5% SDS at room temperature for 30 min and in 0.2x SSC and 0.5% SDS at 65 °C for 30 min. Next, the signals were visualized with a BAS-3000 image-analyzer (GE Healthcare, Little Chalfont, UK).

In-vitro digestion reactions were carried out with three different types of the Cas12a family (LbCas12a, AsCas12a, and FnCas12a were purchased from New England Biolabs Inc. or purified in the lab, Integrated DNA Technologies Inc., and abm®, respectively) and a wide array of modified crRNAs (purchased from DNA Technologies Inc.). Cas12a and crRNA were mixed with a 1:1 ratio (100 nM:100 nM) in 1× NEBuffer 2.1 and pre-incubated at 25 °C for 15 min to promote the ribonucleoprotein complex formation. DNA activator (final concentration of 7 nM) was then added to the mixture to produce a total volume of 30 μl and incubated at 37 °C for 30 min^{19 (link)}. The sample was then analyzed in either 1% agarose gel (for GFP fragments amplified from the pEGFP-C1 plasmid) pre-stained with either SYBER Gold (Invitrogen), GelRed (Biotium Inc.), or premade 15% Novex^TM TBE-Urea Gel (Invitrogen).