Illumina HiSeq 2000 sequencing; TEAD and YAP regulate the enhancer network of human embryonic pancreatic progenitors
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Pancreas
Cell type
Pancreatic bud
NA
NA
Attributes by original data submitter
Sample
ENA first public
2015-04-06
ENA last update
2018-03-08
ENA-CHECKLIST
ERC000011
External Id
SAMEA3121281
INSDC center alias
IDIBAPS and IFIBYNE-CONICET
INSDC center name
IDIBAPS and IFIBYNE-CONICET
INSDC first public
2015-04-06T17:04:46Z
INSDC last update
2018-03-08T21:57:29Z
INSDC status
public
Submitter Id
E-MTAB-3061:PB_FOXA2_rep2
broker name
ArrayExpress
common name
human
developmental stage
Carnegie Stage 16-18
organism part
pancreatic bud
sample name
E-MTAB-3061:PB_FOXA2_rep2
Sequenced DNA Library
library_name
PB_FOXA2_rep2
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Human embryos were collected with informed consent following approval from the North West Regional Ethics Committee, UK (08/H1010/28) following termination of pregnancy and staged immediately by stereomicroscopy according to the Carnegie classification38. The collection, use and storage of material followed guidelines from the UK Polkinghorne Committee, legislation of the Human Tissue Act 2004 and the Codes of Practice of the Human Tissue Authority, UK. The analysis of human embryonic tissue was also approved by the Comitè Ètic d’Investigació Clínica del Centre de Medicina Regenerativa de Barcelona and Departament de Salut, Generalitat de Catalunya. Human embryonic pancreas and liver were dissected at Carnegie Stage (CS) 16-18, which correlates to ~37-45 days post-conception. These stages were the earliest at which the pancreatic epithelial cells could be efficiently dissected away from surrounding mesenchyme with minimal contamination. After isolation, pancreatic and liver tissues were rinsed with phosphate buffered saline 3 times, formaldehyde was added to a final concentration of 1%, and samples were incubated at room temperature for 10 min. Glycine was added to 125 mM for 5 min, tissue was next rinsed 3 times with phosphate-buffered saline containing protease inhibitor cocktail (Roche) at 4ºC, and then snap-frozen and stored at -80ºC. Total embryonic pancreas RNA was extracted from unfixed tissue using Trizol and then treated with DNase I. Either 7 human CS16-18 pancreatic buds, 4 CS16-18 liver buds, or ~10 million cells from a pool of 3 pancreatic progenitor in vitro differentiation experiments, were pooled in 1 mL of lysis buffer containing protease inhibitor cocktail (Roche) and sonicated 10-15 cycles. We verified that a substantial portion of chromatin fragments were in the 200-600 bp range by gel electrophoresis. ChIP was performed with 50-300 µL of sonicated chromatin. Chromatin was diluted with ChIP dilution buffer (0.75% Triton X-100, 140 mM NaCl, 0.1% sodium deoxycholate, 50 mM Hepes at pH8.0, 1 mM EDTA, 1X protease inhibitor cocktail) to achieve a final SDS concentration of 0.2%, pre-cleared with A/G sepharose beads (GEHealthcare) for 1 hour, and incubated overnight at 4ºC with 1-1.5µg of the following antibodies: rabbit anti-H3K27ac (ab-4729), rabbit anti-H3K4me1 (ab-8895), goat anti-Pdx1 (BCBC AB2027, Chris Wright, Vanderbilt University), goat anti-FOXA2 (sc-6554), rabbit anti-GATA6 (sc-9055X), rabbit anti-HNF1β (sc-22840-X), rabbit anti-ONECUT1 (sc-13050), mouse anti-TEAD1 (BD610922) or rabbit anti-YAP (Cell Signaling). Samples were then rotated 2 hours at 4ºC with A/G sepharose beads and then sequentially washed with Low salt wash buffer (1% Triton X-100, 0.1 % SDS, 150 mM NaCl, 2mM EDTA, 20 mM Tris-HCl pH 8.0), High salt wash buffer (1% Triton X-100, 0.1 % SDS, 500 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl pH 8.0), LiCl wash buffer (1% NP40, 0.25 M LiCl, 1 mM EDTA, 1% deoxycholate sodium, 10 mM Tris-HCl pH 8.0), TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Immune complexes were eluted from the beads by adding IP elution buffer (1% SDS, 0.1M NaHCO3). Next, chromatin was sequentially treated with RNAse (greater than 5 hr at 65 degrees C) and Proteinase K (overnight at 45 degrees C). Finally, DNA was extracted with phenol-chloroform followed by ethanol precipitation. For all libraries 5-10 ng of DNA were used to prepare ChIP libraries according to Illumina protocols, and sequencing of ~25 million (for transcription factors and H3K27ac) or ~50 million (for H3K4me1) single-end reads was performed on Illumina HiSeq2000 platform using standard recommended procedures.