Total RNA was isolated using RNeasy Plus Micro Kit (Quiagen) and was quantified and purity checked using a NanoDrop ND-1000 (Thermo Scientific, Waltham, MA, USA). RNA integrity was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) 500 ng of total RNA were used with the TruSeq RNA Sample Preparation v2 Kit (Illumina, San Diego, CA) to construct index-tagged cDNA libraries. Libraries were quantified using a Quant-iT™ dsDNA HS assay with the Q-bit fluorometer (Life Technologies, Carlsbad, California). Average library size and the size distribution were determined using a DNA 1000 assay in an Agilent 2100 Bioanalyzer. Libraries were normalized to 10nM using Tris-Cl 10mM, pH8.5 with 0.1% Tween 20. Libraries were applied to an Illumina flow cell for cluster generation (True Seq SR Cluster Kit V2 cBot) and sequence-by-synthesis single reads of 75 base length using the TruSeq SBS Kit v5 (Illumina) were generated on the Genome Analyzer IIx following the standard RNA sequencing protocol. Reads were further processed using the CASAVA package (Illumina) to split reads according to adapter indexes and produce fastq files. RNA-Seq was performed at the Genomics Unit of the Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid.
GEO Accession
GSM4696960