Transcriptional and epigenetic profiling of the progression of hESCs to beta cells
Transcriptional and epigenetic profiling of the progression of hESCs to beta cells
Strand-specific RNA-seq libraries were prepared as previously described [Parkhomchuk, D., et al., Nucleic Acids Res, 2009. 37:e123], with minor modifications. Following purification, mRNA was primed with Olig(dT)s and random hexamers, and reverse-transcribed to first-strand cDNA. Residual dNTPs were removed from the first-strand cDNA using Illustra MicroSpin G-25 columns (GE Healthcare). In the second-strand synthesis reaction, dUTPs were used instead of dTTPs. The converted double-strand cDNA was fragmented using a Bioruptor Sonicator for 60 cycles of 30 sec on and 30 sec off. After end-repair and adenine base addition, the cleaved double-strand cDNA fragments were ligated to Pair-end Adaptor Oligo Mix (Illumina) and size-fractionated on a 2% high resolution agarose gel (Sigma). cDNA fragments of 200 (+25 bp,-25bp) were recovered, and incubated with uracil-N-glycosylase (UNG) to digest the second-strand cDNA. Purified single-strand cDNA was then used as template for 15 cycles of amplification using pair-end PCR primers (Illumina). The amplified products were separated on a 2% agarose gel and a band between 225-275 bp excised.
Experimental Factor: TIMEPOINT
n/a
Experimental Factor: CD antibody
n/a
Experimental Factor: histone antibody
n/a (rna-seq)
Experimental Factor: CELL_TYPE
islet cell
Experimental Factor: CELL PROPERTY
n/a