Udg enzyme

FFPE-derived DNA was pre-treated to reduce the impact of potential DNA damage, before target capture. 100 ng of DNA, as quantified by the Qubit dsDNA HS kit (ThermoFisher Scientific), was digested with 1 unit of UDG enzyme (New England Biolabs (NEB)) in a 50 μL reaction in 1X of the supplied reaction buffer (NEB). The mixture was incubated at 37°C for 10 minutes, cooled to 4°C, and immediately purified with the Agencourt AmPure XP Kit at a 3:1 bead to sample volumetric ratio as per manufacturer’s instructions. Samples were eluted in 20 μL of 10 mM Tris-HCl, pH 8. Total amplifiable DNA was quantified using KAPA hgDNA Quantification and QC Kit (KAPA Biosystems) as per the manufacturer’s instructions.

RNA in liver tissue was isolated and purified by TRIzol (Invitrogen, CA). After the quality control of total RNA quantity and integrity, the mRNA containing PolyA was specifically captured through two rounds of purification using Oligo (dT) magnetic beads (25–61005, Thermo Fisher, USA). The mRNA was captured under high-temperature conditions for fragmentation. The fragmented RNA was transcribed and U-labeled second-stranded DNAs was synthesized, and digested with UDG enzyme (NEB, m0280, MA, US). PCR was performed to form a library with a fragment size of 300 bp ±50 bp and an Illumina Novaseq ™ 6000 (LC Bio-Technology Co., Ltd. Hangzhou, China) was employed to perform double-ended sequencing according to standard operations, with the sequencing mode being PE150. Fastp was used to perform quality control. HISAT2 was used to compare sequencing data to the genome. StringTie was used to assemble genes or transcripts and quantify them using FPKM. The differentially expressed mRNAs were selected with fold change > 2 or fold change < 0.5 and with parametric F test comparing nested linear models (p < 0.05) by R package edgeR. Data was deposited into the NCBI Sequence Read Archive (SRA) database (Accession Number: PRJNA1045022).

Total RNA was isolated and purified using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s recommended procedure. Poly-(A) RNA was purified from 1 μg total RNA using Dynabeads Oligo-(dT)25-61005 (Thermo Fisher, CA, USA), with two rounds of purification. The poly-(A) RNA was fragmented at 94 °C using the Magnesium RNA Fragmentation Module (NEB, USA). The RNA fragments were reverse transcribed to cDNA using SuperScript II Reverse Transcriptase (Invitrogen). The first-strand cDNA was then used to synthesize U-labeled second-stranded cDNA. An A-base was then added to the blunt ends of each strand to prepare them for the ligation to the index adapters. Single- or dual-index adapters were ligated to the fragments before the size selection step was performed using AMPureXP beads. After the heat-labile UDG enzyme (NEB, USA) treatment of the U-labeled cDNA, the ligated products were amplified by PCR. The average insert size for the final cDNA library was 300 ± 50 bp. Finally, the cDNA library was sequenced (2 × 150-bp paired-end sequencing) on the Illumina NovaSeq 6000 system (LC-Bio, Hangzhou, China) following the vendor’s recommended protocol.