alias: E-MTAB-7185:Diff5_28_S15_s
title: NextSeq 500 sequencing; RNA-seq of Human Embryonic Stem Cell derived pancreas progenitor differentiation, Day 13 of wildtype, HES1-/-, NEUROG3-/- and HES1-/-NEUROG3-/- genotypes
design: RNA-seq of Human Embryonic Stem Cell derived pancreas progenitor differentiation, Day 13 of wildtype, HES1-/-, NEUROG3-/- and HES1-/-NEUROG3-/- genotypes
library_construction_protocol: Washed once in PBS, and then harvested in RLT plus. Seeded each cell line at 150.000cells/cm2 in 12-wells in 1mL  after single cell suspension with TrypLE Select 5 min, spin down and resuspended in media then counting on Sceptor cell counter 40µM (Millipore) diluted 1:10 in PBS. Differentiation experiments were performed three times (Diff #3, #4 and #5), each for all 12 cell lines, harvested for RNA and protein on various days, including for RNA Day 10 and Day 13 for all experiments. For immunocytochemistry staining cell lines H1, 22, 21sub10, 24sub3, H3(4th diff exp.), 101, 14(5th diff. exp.), 12, 28 were seeded in Ibidi slides as explained above.For 48 hours prior to differentiation cells were kept in E8 hESC media changing media every day also for differentiation. The protocol is slightly modified version from the Kieffer Lab (Rezania et al., 2014) including porcupine inhibitor IWP-6 at 5µM for days 3-6. Compounds and solutions are from ThermoFisher unless otherwise stated.  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  For building RNA-seq libraries two cell lines from each genotype and from differentiation #4 and #5 with 1µg 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 and the libraries were loaded accordingly on
identifiers.namespace: Novo Nordisk Foundation Centre for Stem Cell Biology, University of Copenhagen, Denmark
identifiers.id: E-MTAB-7185:Diff5_28_S15_s
attributes.tag: Experimental Factor: genotype
attributes.value: HES1 and NEUROG3 homozygous double knockout