GSM4818023: RS0042; Homo sapiens; RNA-Seq
A blood sample was collected in PAXgene Blood RNA tubes (PreAnalytiX, Hombrechtikon, Switzerland) before the administration of the anti-TNF agent (week 0) and after 2 weeks of treatment (week 2). Total whole blood RNA was extracted using PAXgene Blood RNA kits (PreAnalitiX) according to the manufacturer's instructions. RNA concentrations were measured using the Quawell Q5000 Spectrophotometer (Quawell Technology Inc, San Jose, CA, USA). RNA integrity was verified using the Agilent RNA 6000 Nano Kit in a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Extracted RNA was stored at −80 °C until further processing. All RNA samples fulfilled the following criteria: RNA integrity ˃7. 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. 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 or the HiSeq 4000 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.