GSM7015603: cMyBP-C+/+ ECT 3 [s20020815E7]; Homo sapiens; RNA-Seq
Total RNA was extracted from day-14 ECTs using the Direct-zol™ RNA MiniPrep kit (Zymo Research). Purity and integrity via the NanoDrop One Spectrophotometer and Agilent 2100 Bioanalyzer, respectively. RNA libraries were prepared from samples that met the TruSeq® Stranded Total RNA Sample Preparation Guide (15031048 E) input guidelines using the Illumina® TruSeq® Stranded Total (Gold) RNA Sample Preparation kit (Illumina Inc., San Diego, California, USA). For each library preparation, cytoplasmic ribosomal RNA was removed using biotinylated target-specific oligos combined with paramagnetic beads tagged with streptavidin. Following purification, the reduced RNA was fragmented using divalent cations under elevated temperature. Fragmented RNA was copied into first stranded cDNA using SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, California, USA) and random primers. Second strand cDNA was synthesized using a modified dNTP mix (dTTP replaced with dUTP), DNA Polymerase I, and RNase H. Double-stranded cDNA was cleaned up with AMPure XP Beads (1X) (Agencourt, Beckman Coulter). The cDNA products were incubated with Klenow DNA Polymerase to add a single 'A' nucleotide to the 3' end of the blunt DNA fragments. Unique dual indexes (UDI) were ligated to the DNA fragments and cleaned up with two rounds of AMPure XP beads (0.8X). Adapter ligated DNA was amplified by PCR and cleaned up with AMPure XP beads (0.8X).  Final libraries were assessed for size and quantity using an Agilent DNA1000 chip and Qubit® dsDNA HS Assay Kit (Invitrogen, Carlsbad, California, USA), respectively. Libraries were standardized to 2nM.