title: total RNA Seq of homo sapien: adult left ventricular myocardium (after CPB ischemia): Sample 42V_post
design: Tissue Sample Collection Methods: Whole transcriptome NGS RNA-Sequencing was performed on Left Ventricular (LV) tissue samples obtained from patients undergoing an elective Aortic Valve Replacement (AVR) with use of Aortic Cross-Clamp (AoXC) and CardioPulmonary Bypass (CPB). Paired myocardial punch biopsies (approximately 3 to 5 µg total RNA content) are collected by a single surgeon using a routinely placed surgical vent in the anterolateral apical LV wall, before and after cold cardioplegia induced ischemia (baseline/pre-ischemia and post-ischemia samples respectively).   Tissue samples were immediately placed in RNAlater (Ambion; ThermoFisher Scientific, Waltham, MA) and after 48 h at 4°C, were stored at −80°C until RNA extraction. Total RNA was isolated with TRIzol (Invitrogen, Carlsbad, CA), and RNA quality was assessed using the Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA) with no samples being excluded for poor quality.  Library Preparation Methods BGI: mRNA samples from total RNA were prepared using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA). Oligodeoxythymidine (oligo[dT]) beads isolated poly(A) mRNA, then mRNA fragmentation was performed using divalent cations under elevated temperatures. Using these short fragments as templates, random hexamerprimer were used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized. Short fragments were purified with QiaQuick p extraction kit (Qiagen, Hilden, Germany) and resolved with elution buffer for end reparation and poly(A) addition. Subsequently, the short fragments were connected with sequencing adaptors. For polymerase chain reaction (PCR) amplification, we selected suitable template fragments with respect to gel electrophoresis results. Template molecules were used for cluster generation and sequencing on the Illumina HiSeq 2500 (Illumina, San Diego, CA). All samples were sequenced using 91 base-pair, paired-end sequencing.  Raw reads produced by the Illumina sequencer imaging files were filtered to remove reads containing adaptor sequences, containing more than 5% unknown nucleotides, or having more than 50% of reads with base quality scores less than 5. Files were aligned to reference genome GRCh37 using STAR software v2.5.2b. Analysis was performed on aligned BAM files using Partek® Genomics Suite 6.6 (Partek Inc., St. Louis, MO).