title: EVC protein regulates sonic hedgehog signalling during human intervertebral disc development and degeneration
design: EVC protein regulates sonic hedgehog signalling during human intervertebral disc development and degeneration
library_construction_protocol: A previously described human NP cell line (NP-nR105, PMID: 24972717)  was cultured in DMEM high glucose (Sigma-Aldrich A5796) with 10% fetal bovine serum (Gibco A5256701), 100 units/mL antibiotic antimycotic solution (Sigma-Aldrich A5955), 10 µM ascorbate-2-phosphate (Sigma-Aldrich A8960), and 100 nM sodium pyruvate (Sigma-Aldrich S8636) at 37°C under 5% CO2. Human NP EVC knockout (EVC-/-) cells were generated using CRISPR/Cas9 targeting EVC exon 4 with an sgRNA (TGTCATCGCTGGTGGCCGAG TGG) designed in-house and synthesized by Integrated DNA Technologies (IDT). The sgRNA was delivered via electroporation (1400V, 20ms, 2 pulses). Monoclonal lines were isolated by serial dilution and expanded. Knockout validation was confirmed using Sanger sequencing, IF, and Western blot analysis. To activate the Hedgehog pathway, WT and EVC-/- cells at approximately 80% confluence were switched to 0.5% FBS medium supplemented with 1µM Smoothened agonist (SAG, Calbiochem 56661) for 40 hours. RNA samples from WT and EVC-/- human NP cells with or without SAG treatment (n=4) were prepared using TRIzol.  RNA samples from WT and EVC-/- human NP cells with or without SAG treatment (n=4) were prepared using TRIzol.  Illumina stranded mRNA library