RNA-Seq Library Preparation and Sequencing

The quality of extracted RNA was checked by Agilent TapeStation 42000 on Agilent RNA ScreenTape (Agilent technologies) before proceeding to library preparation. Only samples with a RINe of 7 or higher were used for Library preparation. RNA-seq libraries were generated using the NEBNext Ultra Directional RNA Library Prep kit for Illumina with NEBNext Poly(A) mRNA Magnetic Isolation Module (both New England BioLabs). Samples were adjusted to 400 ng total RNA and spiked with diluted 1/100 ERCC Spike-In Mix (4456740, Invitrogen) and Drosophila melanogaster, embryo Poly A+ RNA, 5 μG (636224-Takara) using 1 μl from each. PCR Enrichment of Adaptor Ligated DNA was run with 12 cycles and SPRI beads (Beckman) were used for library clean up. Agilent DNA Screen tapes (Agilent Technologies) were used to check library size and quality. Samples were sequenced on an Illumina Novaseq 6000 platform with 180 pM loading concentration in single-end run using 100-bp reads. Enriched GO terms (Biological process) were filtered and mapped using the REViGO tool available at http://revigo.irb.hr/ [50 (link)]. The raw RNAseq data from this study can be accessed from ENA via accession PRJEB49943.