Fast cells to ct kit

U2OS cells (one 150 mm dish per biological replicate) were harvested by trypsinization (0.25% trypsin; Life Technologies) and pelleted at 1,000 g for 5 min in 15 mL conical tubes. Cell pellets were washed with PBS (Life Technologies) and divided for RNA, DRIP or LM-PCR harvests. RNA was either purified and retro-transcribed using RNA purification (Qiagen) and SuperScript IV Reverse Transcriptase (Thermo #8090050) kits, respectively, or using Fast Cells-to-CT kit (Thermo #4399003). qPCR was done using the same settings as those for DRIP-qPCR and LM-PCR methods, and GAPDH used as a reference gene.

Astrocytes from two control and two Krabbe donors were thawed and seeded into a poly-L-ornithine (10 μg/mL, Sigma-Aldrich) and laminin (10 μg/mL, Sigma-Aldrich) coated 96 well plate in complete astrocyte medium and cultured for 7 days, with 50% medium changed after 3 days. Astrocyte cultures were treated with 50 ng/mL TNFα (Sigma-Aldrich) diluted in complete astrocyte medium, or mock medium as a control. Cells were incubated with TNFα for 24 hours, following which medium was aspirated and cells were harvested for RNA expression via qPCR using the Fast Cells-to-Ct kit (Thermo-Fisher Scientific) as described below.