Synchronization and Transcriptome Analysis of C. elegans

CX12311, PTM413, and PTM414 were synchronized using alkaline-bleach to isolate embryos, which were washed with M9 buffer and placed on a tube roller overnight. Approximately 400 hatched L1 animals were placed on NGM agar plates for each strain and incubated at 20°C for 48 hours. The ~L4 stage animals were washed off for standard RNA isolation using Trizol. Four replicates for each strain were performed on different days. The RNA libraries were prepared using the NEB Next Ultra II Directional RNA Library Prep Kit (E7760S) following its standard protocol. The libraries were sequenced by Illumina NextSeq 500. The reads were aligned by HISAT2 using default parameters for pair-end sequencing. Transcript abundance was calculated using HTseq and then used as inputs for the SARTools [88 (link), 89 (link)]. edgeR v3.16.5 was used for normalization and differential analysis[55[90 ]. The analysis result was shown in a volcano plot. CX12311 was treated as the wild type. The genes show significant differential expression in the volcano plot are under thresholds | log₂(fold) | > 1 and FDR adjusted p-value < 0.01. Sequencing reads were uploaded to the SRA under BioProject PRJNA526525.