title: GSM5175918: rrf3_mutant_dt7_R1_sRNA-seq; Caenorhabditis elegans; ncRNA-Seq
library_construction_protocol: Synchronous populations of worms were grown at 20°C on NGM plates seeded with OP50 E. coli concentrated food or otherwise described in treatment protocol , at a density of maximum 40,000 animals per 15 cm Petri dish. Worms were carefully monitored using a stereomicroscope and sorted using COPAS bio sorter at 44 h to enrich for late L4 stage larval population. Sorted worms were frozen in dry ice with Trizol reagent (Ambion). After five repetitions of freeze and thaw in dry ice, total RNA was isolated according to the Trizol Reagent protocol. Agilent 2200 TapeStation System was used to evaluate the RIN indexes of all the RNA preps, and only samples with RIN > 8 was used for downstream applications. 6 µg of total RNAs with RIN > 8 was size selected from 15% UREA PAGE gel and used to generate small RNA libraries. The library preparation was performed as essentially described by Jayaprakash et al., 2011, except that a 5' polyphosphatase (Lucigen RP8092H) treatment was performed to be able to clone tri-phosphate small RNAs, and that the PAGE gel extraction after each ligation was substitute with purification by 1.8 volumes of Agencourt RNAClean XP Beads (Beckman Coulter, NC0068576) and 3 volumes of isopropanol. The multiplexed amplified libraries were further purified using PippinPrep DNA size selection with 3% gel cassettes and the following parameters for the selection: BP start (115) and the BP end (165). The purified libraries were quantified using Qubit Fluorometer High Sensitivity dsDNA assay kit (ThermoFisher, Q32851) and sequenced either on NextSeq-500 Illumina platform using the NextSeq 500/550 High Output v2 kit 75 cycles (FC-404-2005).