GSM6601057: C. elegans, no bacteria, 3h, rep1; Caenorhabditis elegans; RNA-Seq
For each time point 10 heads were pooled into a pre-cooled Eppendorf tube on dry ice, containing 500 µl trizol and 1 µl GlycoBlue. Samples were frozen at -80°C for storage and later subjected to RNA sequencing according to the Tomo-Seq protocol (Holler and Junker, 2019) with minor modifications. Briefly, after thawing, samples were mixed thoroughly and incubated for 5 min at room temperature. Subsequently, 100 µl of chloroform were added to each sample, mixed well and incubated for 5 min. Samples were then centrifuged at 12,000 g for 15 min at 4°C. Next, the aqueous phase was carefully transferred to an Eppendorf LoBind containing 250 µl isopropanol and precipitated overnight at -20°C. Samples were centrifuged for 10 min at 12,000 g and at 4°C. Next, the supernatant was removed and the RNA pellet was washed with 75% freshly prepared ethanol. After washing, the pellet was dried and, once completely dry, resuspended in 2 µl molecular grade water. 1 µl of the sample was transferred to a fresh low binding PCR tube containing barcoded oligo(dT) primer. Reverse transcription was performed with the Superscript II kit according to (Holler and Junker, 2019). First strand synthesis was performed for 1 h at 42°C. Second strand synthesis was performed for 1 h at 16°C. Next, samples were pooled and purified with Agencourt AMPureXP beads. IVT was performed for 16h at 37°C. After fragmentation and purification, RNA was reverse transcribed with superscript II and amplified by PCR. Concentration and size distribution of the resulting cDNA were measured with Qubit and Tapestation. mRNA Libraries were sequenced on a NextSeq500 in paired end mode with 150 cycles.