title: FEB01_C4.NRF2KD.3H.HG
organism: Homo sapiens
description: Protocols: HepG2 cells were seeded on a 6-well plate (2x10^5 cells/well). On the day of the treatment, the media was removed and the wells washed twice with 1X DPBS (14190144 Gibco) and 10 µM of SF or control (DMSO) in serum-free DMEM without glucose (Catalogue: 11966025 Gibco) supplemented with 1 mM sodium pyruvate (11360070 Gibco), and 5.5 mM or 25 mM glucose (A2494001 Gibco). For the continuous SF treatment RNA was extracted at 24 h. After 24 h, for all experimental procedures, following the desired time point: 1) Media was removed from six well plate, 2) cells were washed twice with 1XDPBS,  3) Buffer RLT with β-ME (444203 Sigma), to control and sulforaphane treated cells, were lysed directly on the 6-well plate. RNA was then extracted using the RNeasy Qiagen kit (Ref:74004) according to the manufacturer's instructions. RNA abundance and integrity were assessed using the Nanodrop ND-1000 spectrophotometer. RNA samples were quantified using Qubit 4.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with RNA Kit on Agilent 5300 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA). RNA sequencing library preparation was prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina following manufacturer's instructions (NEB, Ipswich, MA, USA). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented. 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 3'ends, and indexed adapter was ligated to cDNA fragments. Limited cycle PCR was used for library amplification. The dUTP incorporated into the cDNA of the second strand enabled its specific degradation to maintain strand specificity. Sequencing libraries were validated using NGS Kit on the Agilent 5300 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA).