ENAapplication/xmlftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR309/095/SRR30962995/SRR30962995_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR309/096/SRR30962996/SRR30962996_2.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR309/095/SRR30962995/SRR30962995_1.fastq.gzftp://ftp.sra.ebi.ac.uk/vol1/fastq/SRR309/096/SRR30962996/SRR30962996_1.fastq.gzprimary200OKGenomicsSun Yat-Sen Universityhttps://www.ebi.ac.uk/ena/browser/view/PRJNA1171951To investigate the differences in gfp pUG RNA during gfp RNAi inheritance across generations in WT, ddx-19(ths636), npp-14(ths673), and glel-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. In this study, sequencing data for each sample were extracted from the raw sequencing data, and separation was performed based on the self-index of each sample (Primer sequences are listed in Supplemental Table S8). We used the following script to separate different samples from the raw data.(https://github.com/WANLAB20192/Code_DDX-19_paper) (see the file: pUG RNA-seq script.zip/1 spilt.py).The subsequent bioinformatics analysis was performed according to the methods described in this paper.ENAfalsepUG RNA sequencing of DDX-19, NPP-14 and GLEL-1 mutant in gfp RNAi inheritance2025-02-122024-10-18PRJNA1171951