GSM9885883: daf-7daf-16_38h_1mM_replicate1; Caenorhabditis elegans; RNA-Seq
Animals were collected in TRI Reagent (ThermoFisher Scientific AM9738). Following five freeze-thaw cycles, 1-bromo-3-chloropropane was added and RNA in the aqueous phase was precipitated with isopropanol for 2 h at -30°C. Samples were centrifuged at 21,000 x g for 30 min at 4°C to pellet RNA. Pellets were washed three times with 75% ethanol and resuspended in nuclease-free water. Samples were checked for quality/quantity on Fragment Analyzer and Qubit prior to library prep. NEBNext rRNA Depletion Kit v2 (NEB # E7400) and NEBNext Ultra II RNA Library Prep Kit for Illumina (Cat# E7765) were used to generate libraries. The resulting library insert size was 200bp-500bp with a median size around 300bp. Libraries were barcoded using unique dual indexing (E6440S) and pooled for sequencing.