RNA-Seq of human fibroblast-like synoviocytes: RA02 treated scramble siRNA
Total RNA was extracted from human fibroblast-like synoviocytes using TRIzol Reagent (Magen). RNA samples were detected based on the A260/A280 absorbance ratio with a Nanodrop ND-2000 system (Thermo Scientific, USA), and the RIN of RNA was determined by an Agilent Bioanalyzer 4150 system (Agilent Technologies, CA, USA).Paired-end libraries were prepared using a ABclonal mRNA-seq Lib Prep Kit (ABclonal, China). The mRNA was purified from 1 g total RNA using oligo(dT) magnetic beads followed by fragmentation carried out using divalent cations at elevatedtemperatures in ABclonal First Strand Synthesis Reaction Buffer. Subsequently, first-strand cDNAswere synthesized with random hexamer primers and Reverse Transcriptase (RNase H) using mRNAfragments as templates, followed by second-strand cDNA synthesis using DNA polymerase I,RNAseH, buffer, and dNTPs. The synthesized double stranded cDNA fragments were then dapterligated for preparation of the paired-end library. Adaptor-ligated cDNA were used for PCRamplification. PCR products were purified (AMPure XP system) and library quality was assessed onan Agilent Bioanalyzer 4150 system. Finally, the library preparations were sequenced on an IlluminaNovaseq 6000 (or MGISEQ-T7) and 150 bp paired-end reads were generated.The data generated from Illumina platform were used for bioinformatics analysis.