PolyA-enriched RNA-seq of primary human hepatocyte spheroids treated with small molecule inhibitors targeting TEAD
PolyA-enriched RNA-seq of primary human hepatocyte spheroids treated with small molecule inhibitors targeting TEAD
Five to ten wells of liver spheroids were collected in 700µL Qiazol (QIAGEN) and frozen at -80°C until RNA extraction. Spheroids were formed by seeding cryopreserved primary human hepatocytes (PHH) of a male donor in ultra-low attachment 96-well plates (Corning) as previously described (Bell, Scientific Reports, 2016).  For spheroid treatments, free fatty acids were conjugated to 10% bovine serum albumin at a molar 1:5 ratio for 2h at 40°C. Formed spheroids were treated with 240μM oleic acid and 240μM palmitic acid along with 100 nM of either TEAD autopalmitoylationp (TEADap, VT-104, 100nM) or TEAD surface inhibitor (TEADsf, 100nM) inhibitors for 5 days. Raw sequencing output was demultiplexed and converted to fastq files by the inbuilt Illumina bcl2fastq tool. Individual lanes were contatenated to one fastq file per sample (cat file1.fastq file2.fastq file3.fastq file4.fastq > file5.fastq). The samples were incubated at room temperature for 5min, before adding 140µL chloroform (Sigma-Aldrich). This mixture was shaken for 15s, incubated for 3min, and centrifuged at 9000 x g and 4°C for 5min. The aqueous phase was carefully removed, and an equal volume of isopropanol added. This mixture was incubated at room temperature for 10min, before centrifugation at 20.000×g and 4°C for 10min. Supernatant was removed and the pellet washed with 70% ethanol, air-dried, and resuspended in water. The isolated RNA was DNnase treated using the Turbo DNnase Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. In brief, 10µg of RNA was treated with 2U Dnase DNase and 40U RNaseOUT (Thermo Fisher Scientific) at 37°C for 30min. DNase-treated RNA was purified using the RNA clean and concentrator kit (Zymo Research). Strand-specific RNA libraries were generated using the NEBNext Ultra II stranded library kit (New England Biolabs) combined with polyA-coupled beads (New England Biolabs) according to the manufacturer's instructions. The library quality was assessed on a Bioanalyzer 2100 device using DNA High Sensitivity chips (Agilent Technologies) and quantified using a KAPA library quantification KIT (Roche).