Small RNA preparation and qPCR

Total RNA extraction from zebrafish embryos

1. Collected embryos at desired stages were washed with clean Holtfreter's solution. 
2. Removed all liquids and added freshly prepared Pronase solution to the embryos, incubated at 28°C for 5-15 min, until all chorion was almost removed.
3. Washed the embryos with clean Holtfreter’s solution and slightly transferred the embryos into an Eppendorf tube, removed most of the liquid from the top.
4. Add 1 ml of TRIzol reagent (Thermo Fisher) per 100 embryos and vortex until all embryos were disrupted and dissolved in the reagent. (Kept samples at -80°C until all samples were collected).
5. Extracted the total RNA according to the manual of TRIzol reagent.
6. The final RNA pellets were dissolved in an appropriate volume of nuclease-free (NF) water.

Deacylation of total RNA

1. Total RNA was deacylated in 100 mM Tris-HCl (pH 9.0) for 30 min at 37°C.
2. Then RNA was purified by addition of 1/10 volume of 3M NaOAc (pH 5.2) and 2.5 volume of ethanol. Precipitated at -20°C overnight.
3. Centrifuged at 12000 rpm for 10 min at 4°C. 
4. Washed the pellet with 75% ethanol and centrifuged at 12000 rpm for 10 min at 4°C.
5. Remove all the supernatant, dry the pellet, and then dissolved it in an appropriated volume of NF water.

3'End phosphate group removal 

1. About 10 μg RNA was treated with T4 Polynucleotide Kinase (NEB, 20 units) at 37°C for 30 min.
2. The RNA was purified by phenol-chloroform extraction, and the pellet was dissolved in NF water.

Methyl-groups removal

1. 5 μg RNA was treated by ALKB protein mixture [wtALKB, 160 pmol; ALKB (D135S), 200 pmol; and ALKB (D135S/L118V), 200 pmol] in reaction buffer [300 mM KCl, 2 mM MgCl2, 50 μM (NH4)2Fe(SO4)2, 300 μM α-ketoglutarate, 2 mM vitamin C, bovine serum albumin (50 μg/ml), 50 mM MES (pH 5.0), 40 U of RNase inhibitor, and 1× protease inhibitor] for 2 hours at room temperature.  

2. RNA was purified by phenol-chloroform extraction, and the pellet was dissolved in NF water.

    

RNA 3'end polyadenylation

1. Purified RNA was treated by E.coli Poly(A) polymerase (NEB, 5 units) at 37°C for 30 min.

2. RNA was purified by phenol-chloroform extraction, and 1 μl Glycoblue (Thermo Fisher) was added to each sample.

    

Reverse transcription and qPCR 

1. Total RNA was then used for synthesizing the first strand cDNA with GoScript RT Mix Oligo (dT) (Promega, A2791). Oligo-dT primer in the kit was used for RT cDNA synthesis. The reverse transcription process took place at 55°C. 
2. cDNA was diluted to 100 μl with double-distilled H2O (ddH2O), and then the same aliquot of each sample was used for qPCR. 
3. qPCR was accomplished with TransStart Top Green qPCR Super Mix (Transgen). qPCR primers were listed in supplementary table S3 part “Specific tRNA and 5'tRFl Primers”. The results of primer specificity test could be found in supplementary figure 4.