title: GSM8526789: control for peak deletion smRNA sequencing rep. 2; Caenorhabditis elegans; ncRNA-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. Small RNAs were enriched using MirVana miRNA Isolation Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. Small RNA-sequencing libraries were prepared using a previously established small RNA cloning protocol(Li, et al. 2020; Pastore, et al. 2021a; Pastore, et al. 2024). To sequence all types of small RNA species, small RNA samples were treated with the polyphosphatase PIR-1 to remove γ and β phosphates from 5'-triphosphorylated RNAs(Li, et al. 2020). To clone csRNAs, small RNA samples were sequentially treated with intestinal alkaline phosphatase (NEB) and Tobacco acid pyrophosphatase(Gu, et al. 2012). The monophosphorylated RNAs were ligated to a 3' adapter (IDT, Supplementary Table S2) using T4 RNA ligase 2 (NEB) at 15 °C overnight. T4 RNA ligase 1 (NEB) was used to ligate the 5' adapter (IDT, Supplementary Table S2). The ligated products were reverse-transcribed into cDNA using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific). The resulting cDNAs were amplified by PCR. Libraries with unique barcodes were pooled and sequenced on the NovaSeq platform (SP 2×50 bp, Illumina).