GSM6364553: Whole worm, wago-1, rep1; Caenorhabditis elegans; ncRNA-Seq
Synchronized populations of worms were grown on 3.5cm OP-50 seeded NGM plates for 58h at 20°C. Worms were collected and washed three times with M9 buffer (22mM KH2PO4, 42mM Na2HPO4, 86mM NaCl) and 100μl (~5,000 worms) of worms were used as starting material. RNA was isolated using Tri Reagent (Molecular Research Centre). Samples were mixed with Tri Reagent in a 1:4 ratio and frozen at -80°C. Samples were vortexed at room temperature for 15min and frozen again at -80°C for 15min. This was repeated for a total of three times. 100μl of chloroform was then added to the samples and centrifuged at 12,000g for 15min at 4°C. The top aqueous phase was transferred to a fresh tube. Phenol:chloroform:isoamyl alcohol (Sigma-Aldrich) was added in a 1:1 ratio and centrifuged at 12,000g for 15min at 4°C. The top aqueous phase was transferred to a fresh tube. 20μg of glycogen (Ambion) and a 1:1 ratio of isopropanol was added to the samples and incubated at -20°C for 30min. Samples were then centrifuged at 16,000g for 30min at 4°C and the supernatant was removed. The pellet was washed with 900μl of 70% ice-cold ethanol for 10min and centrifuged at 16,000g for 10min at 4°C. This was repeated twice. The pellet was then left to air dry for 10min at room temperature and then resuspended in 6-25μl of RNase free water preheated to 70°C. Small RNA libraries were prepared with the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs) following the protocol provided by the manufacturer. 1μg of total RNA or immunoprecipitated RNA was used as starting material. The resulting DNA library was visualized using 8% PAGE and bands corresponding to small RNAs of size range 16-30bp (∼135-150bp on gel) were excised. The DNA was eluted from the excised bands by rotating overnight in 500μl of DNA Gel Elution buffer at room temperature. The DNA was precipitated with 20μg glycogen (Ambion), 50μl of 3M Sodium Acetate pH 5.2 and 1 volume of isopropanol (Sigma-Aldrich) as described above and ultimately resuspended in 12μl of Ultra-Pure water. The library DNA was quantified using a Qubit HS DNA kit (ThermoFisher). Between 12-19 Libraries were pooled in equal amounts and sequenced on a HiSeq 2500 Sequencing System (Illumina).