NextSeq 500 sequencing; RNA-seq of human ES cell lines HUES4 and H1, differentiated for 5 or 7 days towards pancreatic endoderm
RNA-seq of human ES cell lines HUES4 and H1, differentiated for 5 or 7 days towards pancreatic endoderm
Cells were washed once in PBS, and RNA was harvested in RLT plus. Each cell line was seeded at 150.000cells/cm2 in 12-well plates. Differentiation (Rezania et al., 2014) was performed thrice for HUES4 and twice for H1 hESCs. IWP-L6 (50 nM) and/or TGF-b1 (10 ng/ml) was added from day 3 to day 7 and cells were harvested for analysis at day 5 and day 7. RNA was extracted using RNeasy Mini plus kit on a Qiacube (Qiagen). NEBNext Ultra II RNA Library Prep Kit - Module #E7770S NEBNext PolyA mRNA Magnetic Isolation Module #E7490 NEBNext multiplex Oligos for Illumina. RNA-seq libraries were built from 1Âµg of RNA using the NEB NEXT Ultra II with mRNA magnetic isolation module (NEB #E7775 and #E7490) with 5 cycles of amplification. Quality of the RNA and subsequently of the libraries were measured on a Fragment Analyser.