Instrument: AB 5500xl Genetic Analyzer
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Heart samples were homogenized with TRIzol® agent in a TissueLysser LT (Qiagen, UK). All RNA extractions were performed using a PureLink™ Kit according to the manufacturer’s instructions (Ambion Life Technologies, CA). RNA was quantified using a NanoDrop1000 spectrophotometer (Thermo Fisher Scientific, UK) and the purity and integrity of RNA samples was measured using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip kit (Agilent Technologies, Spain). All samples displayed a 260/280 ratio greater than 2.0 and RNA integrity numbers ≥ 9. Total PolyA-RNA samples were used to generate whole transcriptome libraries for sequencing on the SOLiD 5500XL platform, following the manufacturer’s recommendation (Life Technologies, CA). No RNA-spike in controls was used. Amplified cDNA quality was analyzed by the Bioanalyzer 2100 DNA 1000 kit (Agilent Technologies, Spain) and quantified using the Qubit 2.0 Fluorometer (Invitrogen, UK). The whole transcriptome libraries were used for making SOLiD templated beads following the SOLiD Templated Bead Preparation guide. Bead quality was estimated based on WFA (workflow analysis) parameters. The samples were sequenced using the 50625 paired-end protocol, generating 75 nt + 35 nt (Paired-End) + 5 nt (Barcode) sequences. Quality data were measured using software SETS parameters (SOLiD Experimental Tracking System).