Illumina HiSeq 2500 paired end sequencing; GSM1571055: D0_undiff_1_1; Homo sapiens; RNA-Seq

GEO accession GSM1571055 is currently private and is scheduled to be released on Dec 31, 2015.

We discovered induction of circular RNA in human fetal tissues, including the heart. In this study, we were able to recapitulate this induction by in vitro directed differentiation of hESCs to cardiomyocytes, paving the way for future studies into circular RNA regulation. Overall design: We harvested hESCs at sequential stages of differentiation: undifferentiated (day 0), mesoderm (day 2), cardiac progenitor (day 5) and definitive cardiomyocyte (day 14). We performed RNA sequencing in biological triplicate, with 3-8 technical replicates each.

Induction of circular RNA in fetal heart development recapitulated in in vitro differentiation

Submitted by Gene Expression Omnibus on 27-JUN-2015

Between 1-3 million cells per sample were pelleted for each sample. RNA was extracted as per the manufacturer's protocol for the mirVana kit (Life Technologies). After extraction, RNA was quantified using the Quant-iT RNA BR Assay Kit (Life Technologies). RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent) using RNA Nano-chips. Following RNA isolation (mirVana miRNA Isolation Kit, Life Technologies, Inc.), the RNA was quantified (Qubit RNA Assay Kit, Life Technologies, Inc.), and quality controlled (RNA6000 Nano Kit and BioAnalyzer 2100, Agilent) 200 ng to 1000 ngwas used as input for the Illumina TruSeq Stranded Total RNA with Ribo-Zero Gold sample prep kit (Illumina, Inc.) and sequencing libraries were created according to the manufacturer’s protocol. Briefly, first both cytoplasmic and mitochondrial rRNA was removed by selectively hybridizing biotinylated probes to target sequences and using magnetic beads to capture the bound products. Following rRNA removal, the RNA was fragmented and copied into first strand cDNA using random primers and reverse transcriptase. Next, second strand cDNA synthesis was completed using DNA Polymerase I and RNase H. The cDNA was then ligated to Illumina supplied adapters and enriched with PCR to create the final cDNA libraries. The libraries were then pooled and sequenced on a HiSeq 2000 (Illumina, Inc.) instrument as per manufacturer’s instructions. Sequencing was performed up to 2 X 101 cycles.

Induction of circular RNA in fetal heart development recapitulated in in vitro differentiation

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