GSM5831844: Blood_S6_MO; Homo sapiens; RNA-Seq
Total RNA was isolated using RNeasy Micro Kit (Qiagen) 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) Stranded: 200 ng of total RNA were used with NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs) to construct index-tagged cDNA libraries. Libraries were quantified using a Qubit™ dsDNA HS assay with the Qubit 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. Low Input: Low Input RNAs were used to amplify the cDNA using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech-Takara). 1 ng of amplified cDNA was used to generate barcoded libraries using the Nextera XT DNA library preparation kit (Illumina). Libraries were quantified using a Qubit™ dsDNA HS assay with the Qubit 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 (SR Cluster Kit cBot) and sequence-by-synthesis single reads of 60 base length using the SBS Kit (Illumina) were generated on the HiSeq 2500 following the standard sequencing protocol. Reads were further processed using the bcl2fastq 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.