Applied biosystems quantstudio 3 real time pcr machine

Cells were washed once in PBS and harvested following trypsinization and centrifugation. The pelleted cells (with media removed) were frozen immediately on dry ice for at least 15 min. RNA was extracted using a QIAGEN QIAshredder extraction column (Cat# 79654) and RNeasy Plus RNA Extraction Kit (Cat# 74134). cDNA was generated using the TaqMan Reverse Transcriptase Kit according to the manufacturer’s instructions (Cat# N8080234, Applied Biosystems/Thermo Fisher Scientific, Foster City, CA). See Table S2 for qPCR primer sequences. Quantitative PCR reactions were prepared with SYBR Green Master Mix (Cat# 4367659, Life Technologies) and run on an Applied Biosystems QuantStudio 3 real-time PCR machine (Thermo Fisher). Relative transcript levels were calculated using the ΔΔCT method and normalized to the ACTB gene as described (Tarangelo et al., 2018 (link)).

Three hundred thousand cells were seeded into a 6-well plate. The next day, cells were washed twice with ice-cold 1× PBS and scraped to harvest. RNA was extracted using a Qiashredder extraction column (Qiagen; 79654) and the RNeasy Plus RNA Extraction Kit (74134; Qiagen). cDNA was generated using the TaqMan Reverse Transcriptase Kit according to the manufacturer’s instructions (N8080234; Thermo Fisher Scientific). qPCR reactions were prepared with SYBR Green Master Mix (4367659; Life Technologies) and run on an Applied Biosystems QuantStudio 3 real-time PCR machine (Thermo Fisher Scientific). Relative transcript levels were calculated using the ΔΔCT method and normalized to the ACTB gene. The following primers were used to amplify ACTB: forward, 5′-CATGTACGTTGCTATCCAGGC-3′ and reverse, 5′-CTCCTTAATGTCACGCACGAT-3′; the following primers were used to amplify GPX4: forward, 5′-AGACCGAAGTAAACTACACTCAGC-3′ and reverse, 5′-CGGCGAACTCTTTGATCTCT-3′.