GSM8850469: N2_L1_biological_replicate_1, RNA-Seq; Caenorhabditis elegans; RNA-Seq
For each 100 μl of worm pellet, 1 ml of Trizol was added. 5 cycles of freeze‐cracking were carried out, freezing in liquid nitrogen, followed by thawing in a water bath at 37°C. Tubes were vortexed for 30 s on, 30 s off, for 5 min and incubated for 5 min at room temperature. 200 μl chloroform per ml of Trizol were added, tubes were shaked vigorously, incubated 2–3 min at room temperature and centrifuged at full speed for 10 min at 4°C. The top layer was transferred to a new tube, and RNA was precipitated overnight at −20°C with 1 μl glycogen and an equal volume of isopropanol. RNA sequencing of embryos and L1 was carried out at the Oxford Genomics Centre and for the L4 samples was carried out at Novogene, Cambridge. All genotypes where sequences in biological triplicate. For small RNA library preparation, 1-2 µg (see Beltran et al., Nat Ecol Evo 2020 for details) of RNA were incubated for 1 h at 37 °C with RNA 5′ polyphosphatase (Epicentre) at a final concentration of 1 U µl−1 in a total volume of 20 µl to convert 5′-triphosphate 22G-RNAs into clonable 5′-phosphate 22G-RNAs. RNA was phenol–chloroform-extracted and precipitated overnight with 1 µl of glycogen, 1/10 volume 3M AcNA and 3 volumes of 100% ethanol. Small RNA libraries were generated using the Illumina TruSeq Small RNA Library Preparation Kit according to the manufacturer's instructions and gel-purified to size-select 21–23-nucleotide (nt) inserts. The libraries were sequenced on an Illumina HiSeq2500 system.