Mid_1 (CRX1850121; CRA027732)
Following mRNA purification, fragmentation was performed. First-strand cDNA was synthesized with 6-base random hexamer primers. Generation of the second strand employed a reaction mixture containing buffer, dNTPs, RNase H, and DNA polymerase I. Double-stranded cDNA was purified with the QIAQuick PCR Kit (QIAGEN, Hilden, Germany), followed by end repair, addition of a 3' dA overhang, adapter ligation, and size selection via agarose gel electrophoresis. Libraries were generated by PCR amplification of the selected target fragments. Library quantification employed a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), followed by dilution to a working concentration of 1 ng/microL. Analysis of insert fragment size distribution was conducted on an Agilent 2100 Bioanalyzer (Agilent 2100 Bioanalyzer, Agilent Technologies, Santa Clara, CA, USA). Libraries meeting quality thresholds underwent 150-base pair paired-end (PE150) sequencing using an Illumina system (Illumina Inc., San Diego, CA, USA). submitted_instrument_model: Illumina HiSeq X Ten