TRIzol Reagent

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Protocol tips
- For low 260/230 readings the best approach is to try washing sample (precipitation) with ethanol to desalt it.

Publication protocol

RNA extraction was performed using TRIzol reagent (Thermo Scientific). RNA quality and concentration were evaluated spectroscopically using a NanoDrop 2000c instrument (Thermo Scientific). RNA integrity was subsequently analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Only good-quality RNA with integrity numbers >9 was used. Transcriptome-profiling assays were performed using the Affymetrix Human GeneChip 1.0 ST arrays (Affymetrix, Santa Clara, CA, USA). Briefly, 250 ng total RNA was reverse transcribed into cDNA, then transcribed into cRNA and labeled into biotinylated cRNA using the GeneChip WT PLUS Reagent kit (Affymetrix) according to the manufacturer’s protocols (P/N 4425209 Rev.B 05/2009 and P/N 702808 Rev.6). Labeled cRNA products were randomly fragmented and hybridized onto Affymetrix GeneChips. Arrays were washed and stained with the Affymetrix GeneChip WT Terminal Labeling and Hybridization Kit, before being scanned using a GeneChip Scanner 3000 (Affymetrix). Cell intensity files containing hybridization raw signal intensities were imported into the Partek GS software (Partek, St. Louis, MO, USA) using default options. Resulting expression data (transcript cluster level) were imported into R statistical environment for further analysis. Transcript clusters without chromosome location were removed. Quality of the data was assessed through boxplot, relative log expression, and Pearson correlation. Linear Models for Microarray Data from Bioconductor was used to compare transcript cluster expression between different conditions, according to the author’s recommendations (Linear Models for Microarray Data User’s Guide section 9.5) (https://bioconductor.org/). Resulting P values were adjusted for false discovery rate (FDR) with Benjamini and Hochberg’s FDR (28), and transcript clusters with FDR <0.05 and absolute fold change ≥1.5 were considered as significantly differentially expressed and used for further analyses. The Ingenuity Pathway Analysis (IPA) software (Ingenuity Systems, Redwood City, CA, USA; www.ingenuity.com) was used for transcript cluster mapping, which led to the identification of differentially expressed genes (DEGs), and for data mining, including functional analyses and gene network reconstruction. Right-tailed Fisher’s exact test was used to calculate a P value for functional enrichment analysis: threshold, −log(P) >1.301. Microarray expression data are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under the accession number E-MTAB-3487.

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