title: GSM3578031: piwi_CRISPR_line1_F12_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 at a density of maximum 40,000 animals per 15 cm Petri dish and harvested at Young Adult stage at 48 hours post hatching. The harvested animals were washed three times with M9 buffer and 40 µl of worm pellet was frozen in dry ice with TRI Reagent (MRC, Inc.). After five repetitions of freeze and thaw, total RNA was isolated according to the TRI Reagent protocol. Ten micrograms of RNA were treated with 2 U of Turbo DNase (Ambion) at 37°C for 30 minutes followed by phenol-extraction and isopropanol precipitation. 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. 10 µg of total DNase treated RNAs with RIN > 8 was size seleceted 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) or Illumina MiniSeq platform using MiniSeq High Output Reagent Kit 75-cycles (FC-420-1001).