Poly a tail assay kit

Pools of 100–200 embryos synchronized at 60–90 min were collected in Eppendorf tubes and disrupted with Pellet mixer Cordless (Avantor) in 200 μl Trizol. RNA was obtained using a Maxwell 16 LEV simplyRNA kit (Promega). One microgram of total RNA was used for PAT assays as described in Sallés and Strickland (1995) (link). In some experiments, the poly(A) tail assay kit from Thermo Fisher Scientific (Ref. 764551KT) was used with equivalent results. Gene-specific forward oligonucleotides used for PAT assays were as follows: String (5′-CCGAAACGCAAATGCAAAC-3′), Grapes (5′-CATTGCATTGTTTACGAGTACG-3′), CG8180 (5′-CAGCAGCATTAACTGACGAATCGAC-3′), EDTP (5′-GGCTAAACCGCTCTCCTGTTCTCTAG-3′) and lok (5′-CTGCATTTAAACTGGGCTGCTGCTTC-3′). Amplified products were resolved in agarose gels, and the efficiency of polyadenylation was measured as the ratio of polyadenylated (A+) versus non polyadenylated (A−) RNA, quantified using ImageQuant-TL. The data for Atx2-depleted, Tyf-depleted and Dicer-2 null embryos were normalized to the wt control (set to 100%) that was carried in parallel in each experiment.

N2, DG4269 (GFP::MEX-3), and all mex-3 mutant animals were collected and washed in M9 buffer 6 times then frozen in trizol and stored at -80°C. Total RNA was isolated from these animals using phenol-chloroform and isopropanol extraction. For the poly(A) tail assay, a poly(A) tail assay kit (ThermoFisher Scientific cat #: 764551KT) was used following the protocol outlined by the manufacturer. For the tail-specific primer set, a universal reverse primer provided in the kit was used for all the strains. For N2, DG4269, mex-3(spr5), mex-3(spr6), and mex-3(spr10), the forward primer 5´-CTACGCACAACTAACGGAGA-3´ was used. For mex-3(spr9), the forward primer 5´-TCATGTCCTCCCTCAAAGG-3´ was used and for mex-3(spr7), the forward primer 5´-CCCCAATATATATTCCTACAGTAGG-3´ was used. The PCR products were purified using a Zymo Research DNA clean and concentrator kit (cat #: D4034). The PCR products were cloned into a pCR4-TOPO TA vector using a TOPO TA Cloning kit (ThermoFisher Scientific cat #: K4575J10) following the manufacturer´s protocol. Plasmids containing the insert were analyzed using Sanger sequencing.