Rnaeasy mini columns

Total RNA was extracted from NP cells using RNAeasy mini columns (Qiagen, Hilden, Germany). Before elution from the column, RNA was treated with RNase‐free DNase I (Qiagen). The purified, DNA‐free RNA was converted to cDNA using High‐Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, USA). Template cDNA and gene‐specific primers were added to Power SYBR Green Master Mix (Applied Biosystems), and mRNA expression was quantified using the Step One Plus Real‐time PCR System (Applied Biosystems). β‐actin was used to normalize gene expression. Melting curves were analysed to verify the specificity of the RT‐PCR and the absence of primer dimer formation.

Total RNA was extracted using TRIzol (Life technologies), according to the manufacturer’s instructions. Total RNA was purified using RNAeasy mini columns (Qiagen). First-strand cDNA synthesis was performed using the ProtoScript M-MuLV First-Strand cDNA Synthesis Kit (New England Biolabs), and qPCR was performed using the Power SYBR Green PCR Master Mix (Applied Biosystems). The primers used for qPCR analysis are listed in Table 3. Actin mRNA was used to normalize RT-qPCR data.

Total RNA was extracted from NP cells using RNAeasy mini columns (Qiagen). Purified DNA-free RNA was converted to cDNA using EcoDry™ Premix (Clontech). Equal amounts of template cDNA and gene-specific primers were incorporated into a SYBR Green master mixture (Applied Biosystems) and mRNA expression was quantified using the Step One Plus Real-time PCR System (Applied Biosystems). HPRT was used to normalize gene expression. Melting curves were analysed to verify the specificity of the RT-PCR and the absence of primer dimer formation. Each sample was d in duplicate and included a template- free control. All primers used were synthesized by Integrated DNA Technologies, Inc. All quantitative data is represented as mean ± SE, n ≥ 4 independent experiments.

A375 cells were treated with vehicle control (DMSO) or 5 μM or 10 μM TP-472 for 24 h, after which total RNA was extracted for analysis of gene expression on an Illumina HiSeq 2500 system. Total RNA was extracted using TRIzol^® reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s instructions and purified using RNAeasy mini columns (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Finally, mRNA was purified from approximately 500 ng of total RNA using oligo-dT beads and sheared by incubation at 94 °C. Following first-strand synthesis with random primers, second-strand synthesis was performed with dUTP to generate strand-specific libraries. The resulting cDNA libraries were then end-repaired and A-tailed. Adapters were ligated, and second-strand digestion was performed using uracil-DNA-glycosylase. Indexed libraries that met appropriate cutoffs for both were quantified by qRT-PCR using a commercially available kit (KAPA Biosystems, Wilmington, MA, USA). The insert-size distribution was determined using LabChip GX or an Agilent Bioanalyzer. Samples with a yield ≥0.5 ng/μL were sequenced on an Illumina HiSeq 2500 system. Images were converted into nucleotide sequences using the base-calling pipeline RTA 1.18.64.0 and stored in FASTQ format.

RNA extraction was performed using the Qiagen RNAeasy minicolumns, following the protocol provided by the manufacturer (including DNase treatment), and eluting in 10 uL of DNase, RNase free water. RNA concentrations were spot-checked (ThermoFisher, Nanodrop 2000) to ensure the RNA extraction was successful. cDNA was prepared using random primers and qPCR was run using BioRad’s iQ SYBR Green Supermix. For the analysis of EIIIA and EIIIB inclusion by qPCR, samples were extracted using the Qiagen FFPE extraction kit. qPCR was performed on random primed cDNA, as previously described (see SI Table S2 for qPCR primers)^{12 (link)}.

Ovarian cancer cell lines (PA-1 and SK-OV3 cells) were treated with BAY-850 (5 μM) and control for 48 h were used to prepare total RNA for gene-expression analysis on an Illumina HiSeq 2500 system. Total RNA was extracted using TRIzol^® reagent (Invitrogen) according to the manufacturer’s instructions and purified on RNAeasy mini columns (Qiagen) according to the manufacturer’s instructions. Then, mRNA was purified from approximately 300 ng total RNA using oligo-dT beads and sheared by incubation at 94 °C. Following first-strand synthesis with random primers, second-strand synthesis was performed with dUTP to generate strand-specific libraries. The cDNA libraries were then end-repaired and A-tailed. Adapters were ligated, and second-strand digestion was performed using Uracil-DNA-Glycosylase. Indexed libraries that met appropriate cutoffs for both were quantified by qRT-PCR using a commercially available kit (KAPA Biosystems, Wilmington, MA, USA). The insert-size distribution was determined using LabChip GX or an Agilent Bioanalyzer. Samples with a yield ≥ 0.5 ng/μl were used for sequencing on the Illumina HiSeq 2500 system. Images were converted into nucleotide sequences by the base-calling pipeline RTA 1.18.64.0 and stored in FASTQ format.