GSM8881349: M7C16_CM_3; Homo sapiens; RNA-Seq
On day 40, cardiomyocytes were lysed in a solution of 700μL PureLink Lysis buffer containing 7μL Beta-mercaptoethanol. The cell lysis was quickly transferred to -80°C for storage. The RNA purification was done using the PureLinkTM RNA Mini Kit (Thermo 12183025). The DNA was removed using the on-column Ambion PureLink DNase Set (Thermo 12185010). Total eukaryotic RNA isolates are quantified using a fluorimetric RiboGreen assay. Total RNA integrity is assessed using capillary electrophoresis (e.g. Agilent BioAnalyzer 2100), generating an RNA Integrity Number (RIN). For samples to pass the initial QC step, they need to quantify higher than 500 ng and have a RIN of 8 or greater. Total RNA samples are converted to Illumina sequencing libraries using Illumina's TruSeq RNA Sample Preparation Kit (Cat. # RS-122-2001 or RS-122-2002) or Stranded mRNA Sample Preparation Kit (Cat. # RS-122-2101). (Please see www.illumina.com for a detailed list of kit contents and methods). In summary, the mRNA from a normalized input mass of total RNA is isolated using oligo-dT coated magnetic beads, fragmented, and then reverse transcribed into cDNA. The cDNA is blunt-ended, A-tailed, and indexed by ligating molecularly barcoded adaptors. Libraries are amplified using 15 cycles of PCR. The final library size distribution is validated using capillary electrophoresis and quantified using fluorimetry (PicoGreen) and via Q-PCR. Indexed libraries are then normalized, pooled, and size-selected to 320 bp (tight) using the PippinHT instrument.