RNAseq analysis of dose/dose rate response after gamma Co-60 exposure on C elegans larvae with focus on reproductive effects
RNAseq analysis of dose/dose rate response after gamma Co-60 exposure on C elegans larvae with focus on reproductive effects
whole worms from petri dishes, Males were separated from hermaphrodites using 40-µm nylon filters and washed three times with M9 buffer Total RNA was extracted from 70-h post-L1 males (him-8 strain; ≥ 300 animals per biological replicate, three biological replicates per condition) subjected to irradiation (0, 1, 10, and 100 mGy/h), using Direct-zol RNA MicroPrep Kit (Zymo Research, Irvine, CA, USA ) according to the manufacturer's instructions. Males were separated from hermaphrodites using 40-µm nylon filters and washed three times with M9 buffer. The volume was reduced to 150 µL in RNase-free Eppendorf tubes, followed by the addition of 600 µL of Tri Reagent (Zymo Research). Samples were kept on ice before and between homogenization steps using bead beating (⌀ 0.1-0.5 mm) on a FastPrep instrument (10 m/s for 20 seconds, repeated once). Following centrifugation, the supernatant was transferred to new RNase-free tubes, mixed with 750 µL of absolute ethanol, and applied to Zymo-spin IC Columns. On-column DNase I treatment (included in the kit) was performed to remove DNA prior to further purification. RNA concentration (>100 ng/µL), purity (A260/A280 > 1.8; A260/A230 > 2.0), and integrity (RIN > 7) were assessed using Qubit (Thermo Scientific, Waltham, MA, USA), Nanodrop (Thermo Scientific) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively. Library preparation was performed by the Norwegian Sequencing Center (Oslo, Norway) using TruSeqTM stranded RNA-seq paired-end libraries with an average insert size of 350 bp for each biological replicate.