Amersham hybond n nylon membrane

293T cells were transfected with the ZFP3 plasmid (this study) or the parental plasmid. After 48 h, cells were lysed. The protocol from LightShift Chemiluminescent RNA EMSA Kit (Thermo Scientific, Cat# 20158) was followed. The sequence of the biotinylated probe is found in S2 Table. Binding reactions were performed in a total volume of 20 μl. Reaction mixtures were loaded on 5% Mini-Protean TBE native polyacrylamide gels (Bio-Rad, Cat# 4565015). Nucleic acids were transferred onto Amersham Hybond-N+ nylon membrane (Cytiva, Caat# RPN203B) by semi-dry transfer (0.8A, 25V, 15 min) using Trans-blot turbo transfer system (Bio-Rad). Crosslinking (120 J/cm2) was performed with a Stratalinker 2400. RNA was visualized on a Chemidoc Touch (Bio-Rad).

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.

To make the formaldehyde denaturing agarose gel, 0.6 g agarose, 5 mL 10× MOPOS buffer was added into 36 mL RNAse free ddH₂O and boiled in the microwave until it completely dissolved. The boiled gel was cooled down to 60 °C, 9 mL 37% formaldehyde was added, and it was poured into the gel cassette and a comb was placed. After the gel was solidified, the comb was taken off carefully because the agarose gum hole spacers are fragile. Gel electrophoresis was performed with vertical electrophoresis equipment (JunYI, Beijing, China) using 1× MOPS buffer at 120 V for various lengths of time. After electrophoresis, glass plates were removed with care from the glass plate–agarose gel–glass plate sandwich using a plastic plate separation tool. The agarose gel was gently placed in the semidry transfer unit (JunYi, Beijing, China) or Trans-Blot Turbo (Bio-Rad, Hercules, CA, USA) to transfer RNA onto the Amersham Hybond-N+ nylon membrane (Cytiva, Buckinghamshire, UK). Electric current of 1 mA per cm² of membrane was used during transferring process. Different lengths of time were tested. The agarose gel was stained in EtBr solution for 10 min and briefly washed in ddH₂O before taking a picture using the Molecular Imager Gel Doc EX System (NEWBIO Industry, Tianjin, China).