Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Single cardiomyocytes were FACS sorted (SH800, Sony Technologies) or manually picked under a microscope. Cells were placed into 96 well plates in water DNase I and RNase inhibitor. DNase was inactivated by increasing the temperature to 72 degrees Celsius for 3 minutes and then cells were placed on ice. SMARTscribe reverse transcriptase, RNase inhibitor, DTT, dNTP, MgCl2, and custom primers were used to convert the mRNA to cDNA. A custom designed PCR primer was used with Advantage2 taq polymerase and was added to the reverse transcription product and dNTP. This was amplified for 19 cycles and was purified using Ampure XP beads (Beckman-Coulter). 100 pg of total cDNA was added to a tagment DNA buffer and spiked with Tn5 (Epicenter) and incubated for 8 minutes at 55 degrees C, which was then stripped off by adding 0.2% SDS to a concentration of 0.05%. Libraries were enriched using KAPAHiFi with index primers and then multiplexed and sequenced.