Nanodrop one nd one w

RNA was extracted using the TRIzol™ Plus RNA Purification Kit (12183555, Invitrogen, USA) according to the manufacturer's instructions. RNA quality control consisted of spectrophotometry using the Nanodrop One (ND-ONE-W, Thermo Scientific, MA, USA) to determine RNA concentration and purity as well as agarose gel electrophoresis to confirm RNA integrity. A DNase treatment with the TURBO DNA-free™ Kit (AM1907, Ambion, TX, USA) was conducted according to manufacturer’s instructions to remove any contaminating genomic DNA carried over from RNA extraction. Once RNA was free of genomic DNA, First strand cDNA synthesis was conducted with the iScript™ cDNA Synthesis Kit (1708891, BioRad, CA, USA).

Genomic DNA was extracted from frozen tissue with a standard phenol/chloroform extraction. Briefly, tissue samples were fully ground with liquid nitrogen and the nuclei were suspended in extraction buffer (1 M sodium chloride, 100 mM Tris, and 50 mM EDTA [pH 8.0]) containing 2% sodium dodecyl sulfate (SDS) and proteinase K (2 mg/ml final concentration). Suspended nuclei were incubated at 56 °C for 2 h and DNA was first extracted with phenol:chloroform:isoamyl alcohol (25:24:1 volume), then with chloroform:isoamyl alcohol (24:1 volume), and precipitated with 0.7 volumes of isopropyl alcohol at − 20 °C for 40 min. DNA precipitates were washed twice with ice-cold 80% ethanol, collected by centrifugation (12,000 rpm, 15 min, 4 °C), dried under vacuum, and resuspended in 100 μl of EB (10 mM Tris hydrochloride [pH 8.0]) (Qiagen, Hilden, Germany). DNA quantity and quality were assessed using a NanoDrop One (ND-ONE-W; Thermo Fisher Scientific Inc., Waltham, MA) and with 1% agarose gel electrophoresis.