GSM4903801: more_than_50-cell_embryos_sRNA-seq; Caenorhabditis elegans; ncRNA-Seq
Synchronous populations of worms were grown at 20°C on NGM plates seeded with OP50 E. coli concentrated food at a density of maximum 40,000 animals per 15 cm Petri dish. Worms were carefully monitored using a stereomicroscope and bleached shortly after worms started to produce the first embryos. Embryos were fixed using 2% Formaldehyde for 30 minutes and sorted using a MoFlo Astrios Cell Sorter. After sorting, embryos were reverse crosslinked at 70˚C for 30 minutes in RIPA buffer prior to RNA extraction to generate small RNA libraries. The library preparation was performed as essentially described by Jayaprakash et al., 2011, except that a 5' polyphosphatase (Lucigen RP8092H) treatment was performed to be able to clone tri-phosphate small RNAs, and that the PAGE gel extraction after each ligation was substitute with purification by 1.8 volumes of Agencourt RNAClean XP Beads (Beckman Coulter, NC0068576) and 3 volumes of isopropanol. The multiplexed amplified libraries were further purified using PippinPrep DNA size selection with 3% gel cassettes and the following parameters for the selection: BP start (115) and the BP end (165). The purified libraries were quantified using Qubit Fluorometer High Sensitivity dsDNA assay kit (ThermoFisher, Q32851) and sequenced either on NextSeq-500 Illumina platform using the NextSeq 500/550 High Output v2 kit 75 cycles (FC-404-2005).