title: GSM8526779: control smRNA sequencing rep. 2; Caenorhabditis elegans; RNA-Seq
library_construction_protocol: Synchronized adult worms were collected, washed three times with the M9 buffer and once in ddH2O, and resuspended suspended in TRI Reagent (Sigma). Worms were lysed using the Bead Mill 24 homogenizer (Thermo Fisher Scientific) and the lysate was transferred to a clean tube. 0.1 volumes of bromochloropropane (Sigma) were added to the lysis to perform RNA extraction. Isopropanol (Sigma) was then used to precipitate RNA from the aqueous phase. Ribosomal RNAs were depleted using the RNase H-based method(Price, et al. 2023a). 3 μg total RNA was incubated with antisense DNA oligonucleotides targeting C. elegans and E. coli ribosomal RNAs, followed by treatment with Hybridase Thermostable RNase H (Biosearch Technologies) at 45 °C for 30 min. The ribosomal RNA-depleted samples were treated with Turbo DNase (Thermo Fisher Scientific) at 37 °C for 30 min. 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 (NEB), following the manufacturer's instructions. The library samples, each containing unique barcodes, were pooled and sequenced on the HiSeq 4000 platform (2x150 bp, Illumina).