title: Expression profiling of Tdo2 RNAi in C.elegans
design: Expression profiling of Tdo2 RNAi in C.elegans
library_construction_protocol: Nematodes were grown at standard conditions at 20 C. Animals were age-synchronized by hypochlorite treatment, hatched over night in M9 buffer and subsequently cultured on Nematode Growth Medium (NGM) seeded with Escherichia coli strain OP50. On day 1 of adulthood (one day after larval stage L4), animals were transferred to NGM plates containing 5-fluoro-2'deoxy-uridine (FUDR) to prevent the offspring from growing. RNAi experiments were performed on NGM plates containing isopropylthio-β-D-galactoside (IPTG, 15mg/L) and 50μg/ml ampicillin. Plates were seeded with RNAi bacteria. Prior to the experiment, the plates were kept at room temperature for 2 days to allow the production of dsRNA by the bacteria. Plates were produced as fresh as possible (maximum 1 week before start of the experiment). Age-synchronized animals were either directly grown on RNAi food from larval stage L1 onwards or transferred to the RNAi treatment at larval stage L4 (indicated for specific experiments). This depended on a test run to see whether the RNAi treatment effected development. Animals of day 1 of adulthood (day after L4 stage) were transferred to RNAi plates containing FUDR to prevent the growth of the offspring. Animals for RNA isolation were collected on day 4 of adulthood in M9. Only samples from the same run were compared later. Animals were washed a few times and then snap frozen in liquid nitrogen. Total RNA was extracted using TRIzol Reagent (Life Technologies) according to the manufacturer's description. The TruSeq Sample Preparation V2 Kit was used (Illumina) for polyA RNA sequencing.