GSM7140768: control sequencing replicate 2; Caenorhabditis elegans; RNA-Seq
Approximately 50,000 synchronized adult worms were collected with M9 using a glass Pasteur pipette. The worms were washed once with M9, rocked in fresh M9 for 5 min, washed once in ddH2O, and resuspended suspended in 1ml of TRI Reagent (Sigma). Worms were lysed using the Bead Mill 24 homogenizer (Thermo Fisher Scientific) and the lysate was transferred to a clean tube 1.5 ml centrifuge tube. 0.1 volumes of bromochloropropane were added to the mix and shaken by hand for 30 seconds. The mixture was then centrifuged at 15,000g and the aqueous phase was transferred to a fresh 1.5 ml microcentrifuge tube, followed by the addition of 1 volume of isopropanol. The tube was incubated at -20 °C for 1 hour to precipitate RNA and centrifuged for 15 mins at 14,000rpm at 4°C. The supernatant removed by pipetting and the pellet was washed twice by adding 1 mL 75% ethanol. The pellet of RNA was allowed to dry for 3 minutes to remove remaining ethanol, dissolved in ddH2O and stored at -80°C. Ribosomal RNAs were depleted using the RNase H method (Adiconis et al. Nature Methods 2013). 3 μg total RNA were incubated with antisense DNA oligonucleotides targeting C. elegans and E. coli ribosomal RNAs, then treated with Hybridase Thermostable RNase H (Biosearch Technologies) at 45°C for 30 minutes. The rRNA-depleted samples were treated with Turbo DNase (Thermo Fisher Scientific) at 37°C for 30 minutes. RNAs longer than 200 nts were enriched using RNA Clean & Concentrator-5 (Zymo Research). mRNA sequencing libraries were constructed using Ultra II Directional RNA Library Prep Kit for Illumina (NEB), according to the manufacturer's instructions. The library samples with unique barcodes were pooled and sequenced at the Illumina HiSeq 4000 platform (PE 150bp).