TMAO1 (CRX1802425; CRA027009)
RNA extraction was performed using the Trizol reagent kit (Accurate Biology, AG21101) following the manufacturer's instructions. RNA integrity was evaluated using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). mRNA was enriched using poly-T oligo-attached magnetic beads. Then, fragmentation was carried out using divalent cations under elevated temperatures in NEB Next First Strand Synthesis Reaction Buffer, and reverse transcription into cDNA was subsequently conducted to produce sequencing libraries using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, 7530). Index codes were added to attribute sequences to respective samples. PCR amplification was carried out using Phusion High-Fidelity DNA polymerase, universal PCR primers, and Index (X) Primer. The products were purified with the AMPure XP system, and library quality was assessed using the Agilent Bioanalyzer 2100 system (Agilent, USA). The qualified libraries were sequenced on Illumina NovaSeq 6000 platforms with PE150 strategy by Novogene Bioinformatics Technology Co., Ltd (Beijing, China).