title: NextSeq 500 paired end sequencing; Targeted inhibition of ubiquitin signaling reverses the metabolic reprogramming and suppresses Glioblastoma growth
design: Targeted inhibition of ubiquitin signaling reverses the metabolic reprogramming and suppresses Glioblastoma growth
library_construction_protocol: Human glioblastoma cells (U87MG) were obtained from the American Type Culture Collection (Manassas, Va.). Cells were cultured in Dulbeccos Modified Eagle Medium supplemented with 10% fetal bovine serum (Gibco Fetal Bovine Serum South America, Thermo Scientific Fisher-US), 2mM L-glutamine, 50 U/ml penicillin, 50 ?g/ml streptomycin, at 37o C, 5% CO2 and 95% of humidity U87MG glioblastoma cells have been transfected, in biological triplicate, with a siRNA targeting praja2 gene or control siRNA. Cells were collected and homogenized in Trizol Reagent. Total RNA extraction was performed with a RNA Clean and Concentrator Kit (Zymo Research Corp., Irvine, CA, USA), following the manufacturers instructions. RNA purity and integrity were assessed with a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and a 4200 TapeStation instrument (Agilent Technologies, Santa Clara, CA, USA), respectively. For RNA purity, an A260/280 ratio of ?2.0 and a A260/230 ratio of 2.0-2.2 were considered acceptable; for RNA integrity, an RNA Integrity Number (RIN) of 9.0-10.0 has been obtained for all samples, indicating the absence of degradation and high integrity of RNA samples. For a precise estimation of the RNA concentration, a Qubit 2.0 fluorometer assay (Thermo Fisher Scientific, Waltham, MA, USA) has been employed