title: NextSeq 500 sequencing; Genome-wide chromatin binding analysis (ChIP-seq) of HA epitope tagged RFX6 protein in human embryonic stem cell derived pancreatic progenitors
library_construction_protocol: Using CRISPR/Cas9 gene editing, the HA epitope was inserted into the 3' end of the RFX6 gene in H9 hESC. Wild-type and HA epitope tagged H9 embryonic 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 ChIP-seq on differentiation day 8. All tissue culture was carried out in a 5% CO2 air environment at 37oC. Day eight differentiation cultures were harvested and processed for chromatin immunoprecipitation as described in the manufacturer's instructions for the SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) (Cell signaling technology, 9005). Chromatin was immunoprecipitated using rabbit anti-HA immunoglobin (Cell signaling technology, 3724). Library preparation was performed using the NEBNext® UltraTM II DNA Library Prep Kit for Illumina (E7645).