alias: E-MTAB-6626:Sample 5_s
title: Illumina HiSeq 2500 sequencing; Transcriptome analysis of pancreatic progenitors differentiated from H9 human embryonic stem cells and induced pluripotent stem cells derived from a Mitchell-Riley Syndrome patient
design: Transcriptome analysis of pancreatic progenitors differentiated from H9 human embryonic stem cells and induced pluripotent stem cells derived from a Mitchell-Riley Syndrome patient
library_construction_protocol: Pluripotent cell lines were obtained as follows: H9 hESC were purchased from WiCell (WA09) and the hiPSC line MRS2-6 was derived in house from fibroblasts taken from a patient with Mitchell-Riley syndrome, using the CytoTune-iPS 2.0 Sendai Reprogramming Kit (Thermo Fisher Scientific, #A16517) in accordance with the manufacturer's instructions. Human pluripotent stem cells were maintained in mTeSR1 media on Matrigel-coated tissue culture plastic, and differentiated into pancreatic progenitors using the STEMdiff Pancreatic Progenitor kit (STEMCELL Technologies, 05120). Differentiation was carried out according to the manufacturer's instructions with the following modifications: (1) cells were initially seeded into 12 well plates (Corning, 353043) at a density of 10e6 cells/well, and (2) stage 1 was extended to last 3 days - rather than 2 - by repeating the final day's treatment. Samples were harvested for RNA isolation on differentiation days 4, 7, 8 and 12. All tissue culture was carried out in a 5% CO2 air environment at 37oC. RNA was isolated from samples harvested from differentiation cultures using an RNeasy mini kit (Qiagen, cat # 74104). All RNA samples had an RNA integrity number > 9. RNA-seq libraries were generated using the NEBNext Ultra RNA Library Prep Kit, (NEB, E7530L). Messenger RNA was enriched by hybridization to oligo-dT.
identifiers.namespace: Postdoctoral Fellow
identifiers.id: E-MTAB-6626:Sample 5_s