Total rna seq h m r library prep kit for

For the RNA-seq of EECs, total RNA was prepared using an RNeasy Plus Micro Kit (Qiagen, Dusseldorf, Germany). The RNA purity was assessed using a Nano Drop spectrophotometer, the RNA concentration was assessed using a Qubit 3.0 fluorometer and the RNA integrity was assessed using an Agilent 2100 Bioanalyzer. The sequencing library was prepared following the instruction manual of a VAHTS total RNA-seq (H/M/R) Library Prep Kit for Illumina^® (Vazyme, China). To isolate the appropriate cDNA fragment size for sequencing, the library fragments were selected with VAHTSTM DNA Clean Beads. The enzyme UDG was used to digest the second strand of cDNA. PCR amplification was performed, and the products were purified. After cluster generation, the libraries were sequenced on an Illumina Hiseq X10 platform (Illumina, California, USA), and 150-bp paired-end reads were generated. Raw reads in the FASTQ format were first processed using in-house Perl scripts. Then, the processed reads were mapped to the reference genome. The reference genome and gene model annotation files were downloaded directly from the genome website. The reference genome index was built using hisat2-build^{14 (link)}, and paired-end clean reads were aligned to the reference genome using Hisat2 (v2.0.5)¹⁵ .