Novaseq 6000 sequencing platform

The mBMDMs (≈1 × 10⁶ cells per sample) cultured with osteoclastogenic medium with/without rapamycin for 4 days were subjected to transcriptome sequencing and targeted metabolomic analysis. Briefly, for transcriptomic analysis, cells were lysed with the Invitrogen TRIzol Reagent to extract cDNA. The cDNA library was constructed using the SuperScript double‐stranded cDNA synthesis kit (Invitrogen, USA) protocol. Illumina NovaSeq 6000 sequencing platform (Shanghai Applied Protein Technology) was performed for transcriptomic analysis. For targeted metabolomic analysis, samples were mixed with methanol, internal lipid standards and methyl tert‐butyl ether (Aladdin, USA). The mixture was adequately vortexed, sonicated and then kept for 30 min. After that, MS‐grade water (Thermo Fisher, USA) was added, and the mixture was vortexed and centrifuged. The upper organic solvent layer was obtained and dried under nitrogen. For LC‐MS analysis, the samples were re‐dissolved in IPA/CAN (9:1, v/v) solvent and then centrifuged. The analysis was performed on a UHPLC system (Nexera LC‐30A, Shimadzu) coupled with QTRAP MS (6500+, Sciex). The analytes were separated on HILIC (Phenomenex, Luna NH2, 2.0 mm ×100 mm, 3 µm) and C18 column (Phenomenex, Kinetex C18, 2.1 × 100 mm, 2.6 µm).

To further investigate the changes in liver gene expression levels at high concentrations of AFB1, chickens fed with 0 mg/kg (control) and 1.2 mg/kg (TB3) AFB1 were sequenced. The construction and sequencing of the small RNA library in this study were completed by Haplox (Nanjing, China). Firstly, a small RNA of 17-30 nt in length was collected by gel electrophoresis, followed by a linker sequence at the 5’ and 3’ ends of the small RNA, and a DNA fragment of 70–80 nt in length was obtained by RT-PCR, and then passed through a gel electrophoresis. The product was recovered by electrophoresis and purified. The sequencing was finally performed using the Illumina Novaseq 6000 sequencing platform (Illumina, San Diego, CA, USA).
The lncRNA library construction in this study was completed by Novogene (Tianjing, China). A total amount of 3 μg RNA per sample was used as the initial material for the RNA sample preparations. Genomic DNA was removed using DNaseI. Six strand-specific cDNA libraries for paired-end 150 bp sequencing were prepared using the dUTP protocol. The library was sequenced on Illumina’s HiSeq platform.

Total RNA from chest muscle tissues of two breed chickens was extracted using the TRIzol reagent. The RNA quality was detected using NanoDrop^® spectrophotometer and Agilent 2100 Bioanalyzer system. The RNA quality in each sample was measured and listed in S4 Table. After that, the cDNA sequencing libraries were prepared as follows: Enrichment of eukaryote mRNA was achieved by utilizing Oligo (dT) magnetic beads from the entire RNA. Following this, the obtained mRNA underwent fragmentation and reverse transcription to generate cDNA. The cDNA was further subjected to end repair, addition of A-tail, and finally connected to the sequencing adaptors. Next, fragment size selection was performed using AMPure XP beads, followed by PCR enrichment to obtain the ultimate cDNA library. Finally, three replicates of BB-sfc and three replicates of BB-bfc cDNA libraries for chicken muscle samples were constructed and raw paired-end reads were generated using the Illumina NovaSeq 6000 sequencing platform from Appiled Protein Biotechnology Co., Ltd. (Shanghai, China).