GSM7140764: eggd-1 null mutant smRNA sequencing rep. 2; Caenorhabditis elegans; miRNA-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. Small RNAs were enriched using MirVana miRNA Isolation Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. β–elimination was performed as described (Pastore et al., (2021)). Small RNA-sequencing libraries were constructed as described (Tang etal., (2016)). Briefly, Small RNA samples were first treated with the polyphosphatase PIR-1 to remove the γ and β phosphates from 5´-triphosphorylated RNAs 4. The monophosphorylated small RNAs were ligated to a 3' adaptor (5'rAppAGATCGGAAGAGCACACGTCTGAACTCCAGTCA/3ddC/3', IDT) using T4 RNA ligase 2 (NEB) at 15 °C overnight. T4 RNA ligase 1 (NEB) was used to ligate the 5' adaptor (rArCrArCrUrCrUrUrUrCrCrCrUrArCrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrU, IDT). The ligated products were converted to cDNA using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific). The cDNAs were further amplified by PCR, and the Small RNA libraries with unique barcodes were pooled and sequenced at the Illumina Novaseq platform (SP 2 X 50 bp).