GSM1571074: D0_undiff_3_6; Homo sapiens; RNA-Seq
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.
GEO
GSM1571074