title: RNA-Seq for HepaRG cells (non-infected) treated with Ruxolitinib replicate 2
design: HepaRG Cells were washed with PBS, harvested by scrapping and precipitated. The precipitates were stored in RNAlater solution at -80C until further use. Total RNA was extracted from cell samples using Qiagen RNeasy Plus Mini kit following manufacturers instructions (Qiagen, Hilden, Germany). Library Preparation with Stranded Poly A selection and NovaSeq Sequencing. Extracted RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA). Strand-specific RNA sequencing library was prepared by using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina following manufacturers instructions (NEB, Ipswich, MA, USA). Briefly, the enriched RNAs were fragmented for 8 minutes at 94 C. First strand and second strand cDNA were subsequently synthesized. The second strand of cDNA was marked by incorporating dUTP during the synthesis. cDNA fragments were adenylated at 3ends, and indexed adapter was ligated to cDNA fragments. Limited cycle PCR was used for library enrichment. The incorporated dUTP in second strand cDNA quenched the amplification of second strand, which helped to preserve the strand specificity. The sequencing library was validated on the Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA). The sequencing libraries were multiplexed and clustered on the flowcell. After clustering, the flowcell was loaded on the Illumina NovaSeq 6000 instrument according to manufacturers instructions. The samples were sequenced using a 2x150 Pair-End (PE) configuration Raw sequence data (.bcl files) generated from Illumina NovaSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq program version 2.20. One mismatch was allowed for index sequence identification.