Library Quantification and RNA Sequencing

All libraries were initially quantified with the Qubit 4 fluorometer using the Qubit^TM dsDNA BR Assay Kit (Thermo Fisher Scientific). Quality assessment and estimation of each library’s average size was evaluated on the Fragment Analyzer system (Agilent Technologies Inc. Santa Clara, United States), using the DNF-477-0500 kit. To further assess the molarity of each library, quantitative PCR (qPCR) was conducted on a Rotor-Gene Q thermocycler (Qiagen, Hilden, Germany) with the KAPA Library Quantification kit for Illumina sequencing platforms (KAPA BIOSYSTEMS, Woburn, MA, United States). Transcriptome sequencing was performed on a NextSeq500 system (Illumina, San Diego), with a NextSeq 500/550 High Output v2 kit (2 × 75 cycles), according to the manufacturer’s instructions.