RNA-Seq of SMG1 inhibition via SMG1i in human foreskin fibroblast (HFF) and human umbilical vein endothelial cells (HUVEC)
Cells were harvested and lysed by adding 500 µl Invitrogen™ TRIzol™ Reagent (Invitrogen; Cat# 15596018) to each well. HFF (TERT-HFF; Ximbio Cat# 154459) and HUVEC (HUVEC/TERT 2; ATCC Cat# CRL-4053; RRID:CVCL_9Q53) cell lines were maintained at 37°C and 5% CO2 in a humidified incubator in (HFF:) DMEM + 10% FBS, + 1% MEM Sigma, Cat# M7145-100ml) or (HUVEC:) EGM Endothelial Cell Growth Medium BulletKit (Lonza, Cat# CC-3124) supplemented with 10% FBS and 20 µg/ml G418. 5x10^5 cells were seeded in 6-well plates one day before starting the SMG1i treatment. SMG1 inhibitor (SMG1i; DOI: 10.1016/j.bmcl.2012.08.107) dissolved in DMSO was added to a final concentration of 1 µM and cells were harvested 24 hours later. Total RNA was extracted using the Direct-zol RNA MiniPrep kit (Zymo Research; Cat# R2052) including the recommended DNase I treatment according to the manufacturer's instructions. poly(A) mRNA isolation and library preparation was carried out with 600 ng RNA as input using the NEBNext Poly (A) mRNA magnetic isolation module (NEB; Cat# E7490L) and NEBNext Ultra II Directional RNA library preparation kit (NEB; Cat# E7760L) according to the manufacturer's instructions.