F2-tempE
RNA-seq libraries were prepared using standard Illumina protocols in DNase- and RNase-free Eppendorf tubes. Five or four biological replicates were processed per condition, each in a separate tube. For each sample, 50ng of total RNA was used to isolate and enrich polyadenylated mRNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, catalog no. E7490L).Subsequent stepsincluding RNA fragmentation, first- and second-strand cDNA synthesis, end-repair, and adaptor ligationwere performed using the NEBNext Ultra II RNA Library Prep Kit with Sample Purification Beads (New England Biolabs, catalog no. E7775L). Adapter ligation was carried out with the NEBNext Multiplex Oligos for Illumina (New England Biolabs, catalog no. E7600A). Libraries were amplified with 14 cycles of PCR. Library concentrations were quantified using the Qubit dsDNA BR Assay Kit (Invitrogen, catalog no. Q32853). The final libraries were pooled in a single Eppendorf tube, stored at 4C, and shipped for high-throughput sequencing.