title: GSM4818887: CSR-1_degron_CSR-1_rescue_ADH_embryos_rep2_RNA-seq; Caenorhabditis elegans; RNA-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. 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 and the embryos pellet was frozen in dry ice with Trizol Reagent (Ambion). After five repetitions of freeze and thaw, total RNA was isolated according to the Trizol 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.  For RNA-seq on FACS sorted embryos, early embryos were crosslinked with formaldehyde to block cell division and were reverse crosslinked at 70˚C for 30 minutes in RIPA buffer prior to RNA extraction. 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. 100 ng 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 using 2.2 volumes of Agencourt RNAClean XP Beads (Beckman Coulter, NC0068576) following the manufacturer's instructions. 100 ng of Ribosomal-depleted RNAs were then used to generate strand-specific RNA libraries using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (E7760S). Multiplexed RNA libraries were quantified using Qubit Fluorometer High Sensitivity dsDNA assay kit (ThermoFisher, Q32851) and sequenced on NextSeq-500 Illumina platform using the NextSeq 500/550 High Output v2 kit 75 cycles (FC-404-2005).