Polytron pt1200e homogenizer

Cell extracts were harvested by direct addition of QIAzol lysis reagent (79306, Qiagen [WERFEN ESPAÑA, S.A.U. L’Hospitalet de Llobregat, Barcelona]) to the culture plate and incubation for 5 min at room temperature (RT) followed by collection in 1.5 mL eppendorf tubes. RNA was isolated using the miRNeasy mini kit (217004, Qiagen [WERFEN ESPAÑA, S.A.U.]) according to the manufacturer’s instructions. For isolation of RNA from mouse tumor tissue, tumors frozen in RNAlater stabilization solution (AM7020, Thermo Fisher Scientific) were thawed in QIAzol and disrupted using a Polytron PT1200E homogenizer (VWR [Llinars de Vallés, Spain]), before RNA extraction using the RNeasy Plus Mini Kit (74134, Qiagen [WERFEN ESPAÑA, S.A.U.]). Purified RNA was stored at −80°C before sequencing. RNA quality control, library preparation, and sequencing were performed by the CRG Genomics Unit. RNA concentration and quality were assessed by nanodrop and bioanalyzer. An average of 90 million 125-nucleotide paired-end reads were generated for each sample. Raw sequencing data were submitted to the Sequence Read Archive (accession number PRJNA551123).

Total RNA was extracted from mouse heart tissue using TRIzol^® Reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol after tissue was mechanically disrupted using a POLYTRON^® PT 1200 E homogenizer (VWR, Radnor, PA, USA) set at maximum speed. Quantity of RNA was measured spectrophotometrically using a Nanodrop 2000 Spectrophotometer (ThermoFisher, Waltham, MA, USA), and 2 μg of RNA per 20 μL reaction was used to synthesize cDNA via random hexamer priming using a High-Capacity cDNA Reverse Transcription kit from Applied Biosystems (Waltham, MA, USA) according to the manufacturer’s protocol. cDNA was diluted 1:5 in ddH₂O. The quantitative real-time (qRT)-PCR duplex reactions contained 7.5 μL PerfeCTa ToughMix qPCR MasterMix (Quantabio, Beverly, MA, USA), 0.75 μL each of the Taqman gene expression assays for FAM-labeled tafazzin (Mm04239390_g1), spanning exons 2/3, and VIC-labeled β-actin (Mm02619580_g1) (Applied Biosystems, Mississauga, ON, Canada), 5 μL of ddH₂O, and 1 μL of cDNA. Transcript levels were measured on a CFX-96 Connect Real Time qPCR Detection System (BioRad, Hercules, CA, USA). Thermal cycling conditions were 50 °C for 2 min, then 95 °C for 20 s, followed by 40 cycles at 95 °C for 3 s, then 60 °C for 30 s. All measurements were run in duplicate. Tafazzin expression was analyzed using the ΔΔCt method, with the Ct values normalized to β-actin.