Metamorphosing individuals, untreated
Total RNA was extracted using Ambion’s miRVana kit, then small (18-30 nt long) RNAs were purified  on a denaturing 15 % acrylamide gel, from 24 to 69 µg total RNA per sample. RNAs were eluted from the acrylamide bands overnight in 0.3 M NaCl, then ethanol/glycogen-precipitated. Half of each sample was oxidized (12 µL RNA; 8 µL 5X borate buffer (148 mM borax, 148 mM boric acid, ph=8.6); 5 µL freshly prepared 200 mM NaIO4 ; 15 µL water) for 30 minutes at 25 degrees C, then precipitated with sodium acetate, ethanol and glycogen; the other half was not treated. These RNA samples were then ligated to the preadenylated 3 ´ adapter (5 ´-rAppdCdT dGdTdA   dGdGdC dAdCdC dAdTdC dAdAdT ddC-3 ´):  1 µL 100 µM preadenylated 3 ´ adapter  2 µL 10X ligation buffer (500 mM Tris pH=7.5; 100 mM MgCl2 ; 600 µg/mL BSA; 100 mM DTT)  2 µL DMSO  1 µL RNAsin (Promega; 40 U/µL)  2 µL truncated Rnl2 ligase (“Rnl2(1-249”, described in Ho, C. K., Wang, L. K., Lima, C. D. & Shuman, S. (2004) Structure and mechanism of RNA ligase. Structure 12, 327–339)  12 µL RNA were mixed and incubated at 25 degrees C for approximately 24h, then ligation products were   purified on a denaturing 15 % acrylamide gel. RNAs were eluted from the acrylamide bands overnight in 0.3 M NaCl, then ethanol/glycogen-precipitated, and ligated to the 5 ´ adapter (5 ´-rGrUrU rCrArG   rArGrU rUrCrU rArCrA rGrUrC rCrGrA rCrGrA rUrC-3 ´):  100 pmol 5 ´ adapter  2 µL 10X T4 RNA ligase buffer (Ambion)  2 µL DMSO   2 µL RNA ligase (Ambion)  13 µL RNA  were incubated 6h at 25 degrees C, then purified on a denaturing 15 % acrylamide gel. RNAs were eluted from the acrylamide bands overnight in 0.3 M NaCl, then ethanol/glycogen-precipitated, and reverse-transcribed:   20 µL RNA + 1 µL 100 µM RT primer (5 ´-dAdTdT dGdAdT dGdGdT dGdCdC dTdAdC dAdG-3 ´) were denatured 2 min. at 72 degrees C, then annealed 2 min. at room temperature, then left a few minutes on ice  0.84 µL DTT  3.36 µL water  4.2 µL dNTP mix (10 mM each)  8.4 µL 5X first strand buffer (Invitrogen)  were added, then each mix was splitted into two tubes: “+RT” (were 2 µL Superscript III, Invitrogen, were added) and “-RT” (were 2 µL water were added), then the reactions were incubated 1 hour at 50 degrees C, and the enzyme was inactivated 15 min. at 70 degrees C.  2 µL of each reaction were used as templates for a PCR amplification (with 25 pmol of each primer: 5 ´-   dAdAdT dGdAdT dAdCdG dGdCdG dAdCdC dAdCdC dGdAdC dAdGdG dTdTdC dAdGdA dGdTdT   dCdTdA dCdAdG dTdCdC dGdA -3 ´ and 5 ´-dCdAdA dGdCdA dGdAdA dGdAdC dGdGdC dAdTdA   dCdGdA dAdTdT dGdAdT dGdGdT dGdCdC dTdAdC dAdG-3 ´, and one PuReTaq Ready-To-Go PCR bead, GE Healthcare, in 25 µL):  2 min. at 94 degrees C, 5 times (15 s. at 94 degrees C, 30 s. at 54 degrees C, 30 s. at 72 degrees C),   17 times (15 s. at 94 degrees C, 30 s. at 60 degrees C, 30 s. at 72 degrees C), then 2 min. at 72 degrees C.   The RT-PCR products were purified on a 2 % NuSieve GTG agarose gel, and extracted with the following protocol:   the agarose bands were melted in 0.4 M NaCl (100 µL per 100 mg agarose) for 15 min. at 70 degrees C; the solution was extracted with 1 volume of phenol (buffered at pH=7.9) at room temperature, then 1 volume of phenol/chloroform/isoamylalcohol (25/24/1 v/v/v), then 1 volume chloroform/isoamylalcohol (24/1 v/v) then precipitated with 2.5 volumes ethanol.  2 pM solutions of the resulting libraries were seeded on each Solexa lane, at the Deep Sequencing Core   facility of UMass Medical School (Worcester, MA, USA).