GSM9416819: Flow1; Homo sapiens; RNA-Seq
After conditioning, ECs from both flow and static grafts were lysed directly in TRIzol. Each RNA sample represented a pooled lysate from three independent batches derived from one HLHS patients. In total, iPSC-ECs from three HLHS patients were used for both flow and static graft conditions. mRNA is purified, from a normalized input between 10-1000ng of total RNA, using mRNA capture beads prior to fragmentation (Watchmaker mRNA Library Prep Kit).  Following first-strand synthesis with random primers, second strand synthesis and A-tailing are performed with dUTP for generating strand-specific sequencing libraries. Adapter ligation with 3' T overhangs are ligated to library insert fragments.  Library amplification amplifies fragments carrying the appropriate adapter sequences at both ends.  Strands marked with dUTP are not amplified. Indexed libraries that meet appropriate cut-offs for both are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution is determined with the LabChip GX or Agilent TapeStation. Samples with a yield of ≥0.5 ng/ul are used for sequencing.