title: NextSeq 500 sequencing; HES1 ChIP-seq in human embryonic stem cell derived pancreas progenitors Day 13.
design: HES1 ChIP-seq in human embryonic stem cell derived pancreas progenitors Day 13.
library_construction_protocol: For Chromatin immunoprecipitation with Next Generation Sequencing (ChIP-seq) we used the HUES4 PDX-GFP reporter human embryonic stem cell line (kind gift from H. Semb and J. Ameri). The reporter cell line was seeded on Fibronectin at cells/cm2 and followed the differentiation protocol as described above. On Day 13 cells were dissociated with TrypLE and  Accutase(ThermoFisher) mix 1:1 for 15 minutes to single cell suspension, spun down 5 minutes 500g and resuspended in FACS buffer (PBS with 10% BSA). Around one quarter was used as an unsorted sample, for the rest added DAPI for live/dead stain and set gates according to MEF GFP-  population on BD ARIA II for sorting. Recovered ~3 million PDX-GFP+ cells per experiment from the sorting and treated them in parallel with the unsorted as follows: spun down 500g for 5 minutes with low acceleration/deceleration and resuspended pelleted cells in 1% Formaldhyde in PBS (freshly made from Amersham MeOH-free 16% Formaldehyde) for 10 minutes. Stopped fixation in 0.125M Glycine for 5 minutes and washed twice in PBS. Then did nuclear enrichment in 0.5% SDS ChIP buffer for 5 minutes and discarded supernatant after spinning.      Lysed in RIPA buffer with protease inhibitors for 10 minutes on ice. Sonicated 7x30seconds ON/OFF, then span full speed for 20 minutes. For the PDX-GFP+ samples 2.5µg chromatin per ChIP and Unsorted 10µg chromatin per ChIP was diluted to 500µL in RIPA buffer. Tumbled overnight at 4°C with primary antibodies 1µL HES1 (H140-X / SC-25392X) and 1µL rabbit IgG (#2729, Cell Signaling). Added Protein G Dynabeads and tumbled for 4 hours, washed in RIPA + inhibitors twice, then once in TE buffer moving to a fresh eppendorff tube and eluted in Elution buffer containing 1%NaHCO3, 1% SDS and proteinase K in PBS for 2h at 56°C and then overnight at 65°C. Cleaned up ChIP'ed DNA with ChIP Clean and Concentrator kit (Zymo) and quantified on a Qubit (ThermoFisher) for sequencing. Built libraries from ~1500pg ChIP'ed DNA for multiplex libraries using a protocol developed in the Ido Amit lab (Blecher-Gonen et al., 2013) and sequenced on an Illumina HiSeq 2500 at the Weizmann Institute, Israel.