small RNA sequencing of DDX-19/NPP-14/GLE-1 mutant

To investigate DDX-19, NPP-14, and GLE-1 regulated small RNAs, approximately 8,000 N2, ddx-19(ths636), npp-14(ths673), and gle-1(ths1041) young adult animals were collected from a 9 cm growth plate using 2 ml of M9 buffer, and transferred into a 1.5 ml Eppendorf tube. After allowing the sample to stand for 1 minute, the supernatant was removed. The animals were washed three times with M9 buffer, with each wash lasting 1 minute. Next, 500 ul of TRIzol Reagent (Thermo Fisher, 15596026) was added to the sample, which was flash-frozen in liquid nitrogen and stored at -80 degrees celsius. When ready to proceed, the samples were thawed on ice, 100 ul of chloroform was added, and they were shaken for 30 seconds before sitting at room temperature for 5 minutes.The sample was centrifuged at 18,000 x g for 10 minutes, and the supernatant was carefully transferred to a new tube. An equal volume of 2-propanol was added, and the tube was inverted and gently shaken 10 times before being precipitated at -20 degrees celsius for 30 minutes. After centrifugation at 18,000 x g for 15 minutes, the supernatant was discarded, and 500 ul of 85% ethanol was added. The sample was mixed thoroughly and spun at 6,000 x g for 5 minutes. The supernatant was removed completely, and the pellet was air-dried at room temperature for 10 minutes. The pellet was resuspended in RNase-free water (Takara, 9012) and stored at -80 degrees celsius.For small RNA sequencing, RNA 5' Polyphosphatase (LGC, RP8092H) was used to dephosphorylate tri- and di-phosphorylated RNA molecules. The following components were combined: 2 ul of 10X Reaction Buffer, 4.5 ug of RNA sample, 1 ul of RNA 5' Polyphosphatase, and RNase-free water to bring the total reaction volume to 20 ul. Each sample was prepared in duplicate. The reaction mixture was incubated at 37 degrees celsius for 30 minutes, and the treated RNA was purified using the method described above. 1 ug of treated RNA from each sample was used to construct small RNA libraries using the VAHTS Small RNA Library Prep Kit for Illumina V2 (Vazyme, NR811-01) following the vendor's protocol. The libraries, containing cloned small RNAs, were run on a 6% Native TBE gel and size-selected according to the Vazyme protocol. The purified, size-selected libraries were sequenced on the Illumina NovaSeq X Plus at HaploX (Shenzhen, China). Each sample had two independent biological replicates.