GSM6166105: C. briggsae smRNA sequencing prg-1 replicate 1; Caenorhabditis briggsae; ncRNA-Seq
Approximately 100,000 synchronized L1 animals were plated on 135 mm NGM plates seeded with OP50 and grown at 20 °C until the animals reached gravid adult stage. Gravid adult populations were harvested using M9, and subsequently suspended in Trizol. Worms were lysed using the Fisherbrand Bead Mill 24 homogenizer, bromochloropropane was added to the lysis to perform RNA extraction. Isopropanol was then used to precipitate RNA from the aqueous phase. Small RNAs were isolated from total RNAs using the miRVana miRNA isolation kit (Thermo Fisher Scientific) according to the manufacturers protocol. Small RNA samples from wild-type and mutants were first incubated with a recombinant 5' polyphosphatase PIR-1 which removes the γ and β phosphates from 5´-triphosphorylated RNAs (Li et al. 2020; Pastore et al. 2021). The resulting monophosphorylated RNAs were ligated to the 3' adaptor (5'rAppAGATCGGAAGAGCACACGTCTGAACTCCAGTCA/3ddC/3', IDT) using T4 RNA ligase 2 in the presence of 25% PEG8000 (NEB) at 15 °C overnight. The 5' adaptor (rArCrArCrUrCrUrUrUrCrCrCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrU, IDT) was then ligated to the product using T4 RNA ligase 1 (NEB) at 15 °C for 4 hours. The ligated products were converted to cDNA using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific). The cDNAs were amplified by PCR using Q5 High-Fidelity DNA polymerase (NEB), and the libraries were sequenced on an Illumina Novaseq platform (SP 2 X 50 bp) at the OSU Comprehensive Cancer Center genomics core.