Illumina NovaSeq 6000 paired end sequencing; Transcriptome analysis of pancreatic endoderm differentiated from induced pluripotent stem cells derived from a patient with Mitchell-Riley Syndrome and her unaffected father
Transcriptome analysis of pancreatic endoderm differentiated from induced pluripotent stem cells derived from a patient with Mitchell-Riley Syndrome and her unaffected father
The human iPSC lines F14, F18, MRS2-6 and MRS2-10 were derived in house from fibroblasts acquired by skin punch biopsy, 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 day 8. 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 > 8. RNA-seq libraries were generated using the NEBNext Ultra RNA Library Prep Kit, (NEB, E7530L). Messenger RNA was enriched by hybridization to oligo-dT.