GSM4818860: CSR-1_degron_CSR-1_rescue_ADH_embryos_rep1_Deg-seq; Caenorhabditis elegans; OTHER
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. Worms were carefully monitored using a stereomicroscope and bleached shortly after worms started to produce the first embryos. The harvested early embryos were washed three times with M9 buffer embryo and the embryos pellet was 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. DNase-treated total RNA with RIN > 8 was used to prepare strand-specific RNA libraries.  We developed an RNase H based method to degrade C. elegans and mitochondrial ribosomal RNAs (rRNAs) using 50nt oligos complementary to rRNA and mtRNA C. elegans sequences. 1 mg of DNase-treated total RNA was mixed with 1.5 µg of oligos at equimolar concentration and 1X probe hybridization buffer (200 mM NaCl, 10 mM Tris pH 7.5) and incubated in a thermocycler using the following parameters: 2 minutes at 95°C followed by 0.1°C/sec at 95-45°C, and 2 minutes hold at 45°C. Next 2 µl of Thermostable RNase H (epicentre) was added to the reactions together with 1X RNase H reaction buffer 50 mM Tris pH 7.5, 100 mM NaCl, 10 mM MgCl2) and incubated for 30 minutes at 45°C. Next, digested RNAs were treated with 2 U of Turbo DNase (Ambion) at 37°C for 30 minutes followed by purification of >200nt RNA molecules using Zymo RNA Clean & Concentrator-5 (Zymo Research: Cat R1015). 5' end adapter was ligated to 5' monophosphate RNA molecules using T4RNA ligase I (Ambion) and incubated at 25 °C for 2h followed by another round of purification of >200nt RNA molecules using Zymo RNA Clean & Concentrator-5. cDNA was synthesized using random hexamer and Superscript IV reverse transcriptase (Thermo Fisher) followed by a first round of PCR amplification using Q5® High-Fidelity DNA Polymerase (New England Biolabs). PCR products >200nt were enriched using 1.8X volumes of AMPure XP for PCR Purification (Beckman Coulter) followed by a second round of PCR amplification. PCR products ranging from 250-450bp were purified on 1% agarose gel and eluted with Qiagen gel extraction kit. 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).