2100 bioanalyzer rna 6000 nano assay

The total RNA was extracted from liver tissue using TRIzol reagent (Invitrogen, Thermo Scientific, MA, USA) following the manufacturer’s instructions. The quantity and quality of RNA were determined using a NanoDrop 2000 ultramicro-spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA). The RNA integrity was estimated using an RNA 6000 Nano Bioanalyzer 2100 Assay (Agilent, Palo Alto, CA, USA). Library construction and sequencing were performed with the Illumina HiSeq platform by Majorbio Biopharm Technology (Shanghai, China).
Expression profiles were obtained using the free online platform of Majorbio Cloud Platform (www.majorbio.com) (accessed on 20 January 2021). In brief, the fold changes were estimated according to the fragments per kilobases per million reads (FPKM) in each sample, and differential expression analysis was performed with the DESeq2 package. We considered differentially expressed genes (DEGs) with p < 0.05 and |log2 FC| > 1. Finally, enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.

Whole blood samples were stored at −80 °C in TRIzol Reagent (Invitrogen, Waltham, MA, USA) until required. Total RNA was obtained from whole blood using the phenol–chloroform method, followed by purification using the RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. To ensure RNA quality, a DNase I treatment was performed using the RNase-Free DNase Set (Qiagen) for 15 min at room temperature. Total RNA extracted from whole blood was sent to the Centre Nacional d’Anàlisi Genòmica (CNAG; Barcelona, Spain) for sequencing. RNA was quantified with a Qubit RNA BR Assay kit (Thermo Fisher Scientific), and the RNA integrity was estimated using the RNA 6000 Nano Bioanalyzer 2100 Assay (Agilent, Santa Clara, CA, USA). The RNASeq libraries were prepared using the KAPA mRNA HyperPrep Kit (Roche, Basel, Switzerland), following the manufacturer’s recommendations, starting with 500 ng of total RNA as the input material. The quality of the library was controlled on an Agilent 2100 Bioanalyzer with the DNA 7500 assay. The libraries were sequenced on NovaSeq 6000 (Illumina, San Diego, CA, USA) with a 2 × 51 bp read length, following the manufacturer’s indications for dual indexing. Image analysis, base calling and quality scoring of the run were obtained by using the manufacturer’s software, Real-Time Analysis (RTA 3.4.4).

Stranded mRNA library preparation and sequencing were performed in the CNAG-CRG platform. The quantity and quality of the total RNA sample determined by Qubit RNA BR Assay kit (Thermo Fisher Scientific) and RNA 6000 Nano Bioanalyzer 2100 Assay (Agilent). The RNA-Seq libraries were prepared with KAPA RNA HyperPrep Kit with RiboErase (Roche) following the manufacturer´s recommendations using Illumina platform compatible adaptors with unique dual indexes and unique molecular identifiers (Integrated DNA Technologies). The final library was validated on an Agilent 2100 Bioanalyzer with the DNA 7500 assay. The libraries were sequenced on NovaSeq 6000 (Illumina) with a read length of 2 × 51 bp following the manufacturer’s protocol for dual indexing. Image analysis, base calling, and quality scoring of the run were processed using the manufacturer’s software Real Time Analysis (RTA v3.4.4) and followed by generation of FASTQ sequence files.