title: total RNA Seq of homo sapien: adult left ventricular myocardium (after CPB ischemia): Sample 4V_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: Harvard Biopolymers Facility We used an approach to construct RNA-sequencing libraries that contains directional cDNA and yields an increased proportion of 5' end sequences. These steps are adapted from published methodologies1,2 and notably do not include normalization or fragmentation. We performed two-rounds of selecting RNAs possessing poly-A tails by annealing them to poly-T oligos bound to beads (Invitrogen, Life Technologies, Grand Island, NY) so as to remove ribosomal RNA. After the RNAs were annealed to the poly-T-beads, unbound material was washed. The polyA species were eluted by melting the annealed structures at high temperature. After 2 rounds of polyA selection, the ribosomal RNA was ~5% of total RNA. Messenger RNAs (mRNAs) were reverse transcribed using random hexamers (Invitrogen). Double-stranded DNA (dsDNA) synthesis was performed using Pol I and RNAse H. End-repair was performed using enzymes such as polymerases and exonucleases to obtain dsDNA fragments with no overhangs. Adenosine addition at 3' ends of both DNA strands was performed with subsequent ligation of appropriate next-generation sequencing adapters. Adenosine overhangs at 3' ends prevent self-ligation of dsDNA fragments during adapter ligation. Size selection was performed by fractionating the DNA library in an agarose gel to excise a size of about ~150-400bp. Size selection allows for appropriate size molecules for next-generation sequencing and enriches fragments that resulted from annealing of random hexamers near the 5' ends of transcripts. Uniform amplification of the library with polymerase chain reaction (PCR) was performed, stopping at the stage of exponential amplification. A library constructed and sequenced with this approach contains directional information owing to the polarity of the complementary DNA (cDNA) molecule.  As samples were analyzed at different times, different read lengths were used, initially using single-end reads (20 samples; 10 subjects) and then transitioning to paired-end reads (188 samples; 94 subjects) ranging from 36 to 100 base pairs. All samples were sequenced on the Illumina HiSeq 2500. 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 UCSC Hg19 using STAR software v2.5.2b. Analysis was performed on aligned BAM files using Partek® Genomics Suite 6.6 (Partek Inc., St. Louis, MO).