ScriptSeq Complete Gold Kit (Epidemiology)

Protocol tips

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	RNA input: 500 ng- 2.5 ug

Protocol tips
RNA input: 500 ng- 2.5 ug

Publication protocol

We performed RNA-sequencing for the differentiated and undifferentiated SH-SY5Y cells. Two µg of
total RNA each sample was subject to RNA-seq library preparation with ScriptSeq™ Complete Gold
Kit from Epicentre (Cat. # SCL24EP, Madison, WI) following the manufacturer’s instructions. In brief,
ribosomal RNA was depleted from total RNA using Ribo-Zero magnetic beads, then the ribosomal RNA
depleted RNA was purified and fragmented. Random primer tailed with Illumina adaptor was used to
perform reverse transcription to get cDNA library. Adaptor sequence was added to the other end of
cDNA library with a Terminal-Tagging step. cDNA library was amplified with Illumina primers
provided with this kit. The product was size selected (350~500 bp) from 2% agarose E-gels (Invitrogen)
and sequenced in one lane per sample on Illumina’s HiSeq 2000 platform. We sequenced two replicates
for each of the differentiated and undifferentiated SH-SY5Y cells, and used cuffdiff (v.2.0.2) to identify
the differentially expressed genes upon the induced differentiation. 258 genes with q value less than 0.05
and fold change (after differentiation) great than 2 were examined for their functional enrichment.
DAVID (http://david.abcc.ncifcrf.gov) was then used to examine the gene ontology enrichment. The
significantly up-regulated genes in the differentiated state displayed GO functional enrichment (by
DAVID) for neuron differentiation (FDR=2.2e-3), neuron projection morphogenesis (FDR=3.2e-3) and
development (FDR=4.1e-3), axonogenesis (FDR=6.2e-3) and synaptic transmission (FDR=4.6e-3),
demonstrating neuron-like characteristics of these cells

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Manufacturer protocol

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