pUG RNA sequencing of DDX-19, NPP-14 and GLE-1 mutant in gfp RNAi inheritance

To investigate the differences in gfp pUG RNA during gfp RNAi inheritance across generations in WT, ddx-19(ths636), npp-14(ths673), and gle-1(ths1041) animals, we conducted gfp RNAi experiments in animals expressing gfp::h2b in the germline. Progeny from the F1, F6, F9, and F15 generations were collected, and total RNA was extracted using TRIzol reagent. Reverse transcription was performed using 5 ug of total RNA and 1 pmol of a DNA oligo containing (AC)9 plus adaptors, utilizing the RevertAid First-Strand cDNA Synthesis Kit (ThermoFisher, K1622) to generate pUG cDNA. Subsequently, three rounds of PCR were conducted. In the first round, 1 ul of pUG cDNA was used in a 20 ul reaction mix containing the initial gene-specific primer and the first adaptor-specific primer. The second PCR round involved a 100-fold dilution of the first-round products, with 1 ul of the diluted sample added to a 50 ul reaction mix containing the second gene-specific primer and the second adaptor-specific primer, as well as sequencing primers for reads 1 and 2, self-indexes for sample identification, and unique molecular identifiers (UMIs, represented as NNN). Following another 100-fold dilution, the third PCR round was performed with primers incorporating Illumina p5 and p7 sequences, along with a unique index in the reverse primer for multiplexing.The final PCR products were pooled, resolved on an agarose gel, and purified. The purified DNA libraries were sequenced on a NovaSeq X Plus platform (Sequanta, China), generating paired-end 150 bp reads.