title: GSM8526697: DHPS-knockdown spheroids supplemented with aspartate, replicate 3; Mus musculus; RNA-Seq
library_construction_protocol: Spheroids were collected and washed in ice-cold cycloheximide-supplemented PBS, lysed in hypotonic buffer (2.5mM MgCl2, 5mM TRIS pH 7.5, 1.5mM KCl supplemented with RNAse and protease inhibitors), and permeabilized by sequential addition of 0.5% Triton X-100 and 0.5% SDS. Lysates were then centrifuged for 7 minutes at 16,000 x g. For each condition, supernatants containing the cytosolic lysates were immediately lysed in Trizol (TriPure Isolation Reagent), snap-frozen in liquid nitrogen, and kept at -80°C for RNA extraction and mRNA library preparation. mRNA library preparation was performed using the Illumina Stranded mRNA Prep kit (ref. 20040534).