GSM9598192: N2_WT_sRNA_R2; Caenorhabditis elegans; ncRNA-Seq
RNA extraction: 3 freeze/thaw cycles - -80 ˚C for 30 minutes followed by 15 minutes vortex at room temperature. 1 volume of chloroform was added to 5 volumes of Trizol+gonads. The mix was put in a pre-spun 2mL Heavy Phase-Lock tube and centrifuged at 16000 g for 5 minutes at 4 ˚C. The aqueous phase was transferred to new pre-spun 2mL Heavy Phase-Lock tube, and 1 volume of Phenol:Chloroform:Isoamyl alcohol (sigma) was added per 1 volume of aqueous phase. The tube was centrifuged at 16000 g at room temperature. Aqueous phase was transferred to Eppendorf tube and precipitated with 1 volume of isopropanol and 1.3 μL glycogen (20 μg/μL). the tubes were put for 30 minutes at -20 ˚C before centrifugation at 16000 g at 4˚C. Pellet was washed with 900μL of cold 70% EtOH, and left at room temperature for 20 minutes before being left at -20˚C over night. The next morning, the tube was centrifuged at 16000 g for 10 minutes, and the pellet was washed again with cold 70% EtOH. Tubes were centrifuged at 16000 g for 10 minutes, all EtOH was removed and pellet was resuspended in 12 μL of warm (70˚C) ddH2O. RNA concentration was determined using Qbit and RNA quality was tested using Agilent 2200 TapeStation. The NEBNext® Multiplex Small RNA Library Prep Set for Illumina® from New England Biolabs® was used for small RNA library preparation. The NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina® from New England Biolabs® was used for mRNA library preparation.