title: GSM9204650: N2 wildtype adult day 2 animals on EV RNAi, treated ad libitum for 24 hours, Replicate 2; Caenorhabditis elegans; RNA-Seq
library_construction_protocol: All RNA-sequencing data pro-processing, quantification, and statistical testing was performed as previously described. Briefly, ~1000 synchronized D1 WT and H3K27ac(-) nematodes were fed ad libitum or fasted for 24 hours and subsequently collected into 500 μl RNAzol (Sigma- Aldrich, Cat# R4533) after 3x washing for total RNA extraction in biologically independent quadruplicates (16 total samples). The total RNA was extracted, and the genomic DNA was removed using the Direct-zol RNA Miniprep Plus Kit (ZYMO research, Cat# R2072) following the manufacturer's instructions. The extracted total RNA was evaluated for quality control using a NanoDrop One C Microvolume UV-Vis Spectrophotometer (Thermo Fisher Scientific), with an A260/A280 > 1.98 and an A260/A230 > 1.85. Samples were sent to Azenta (Genewiz) for additional quality control, library preparation, and mRNA sequencing. Samples were first validated for RNA integrity with a RIN score > 8.5 and DV200 > 50 using an RNA Tapestation 4200 (Agilent). Illumina library preparation was performed using polyA selection for mRNA species. Approximately 17.5 - 30 million paired-end 150bp reads were generated per sample, with > 90% of bases passing a Phred quality score Q30.