characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2015-04-06T17:04:46Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T21:57:29Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA3121279
characteristics.characteristic: External Id
characteristics.text: IDIBAPS and IFIBYNE-CONICET
characteristics.characteristic: INSDC center name
characteristics.text: 2015-04-06T17:04:46Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T21:57:29Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS605429
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-3061:LB_FOXA2
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: FOXA2 ChIP-seq Protocols: 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.
characteristics.characteristic: description
characteristics.text: Carnegie Stage 16-18
characteristics.characteristic: developmental stage
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: liver bud
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: LB_FOXA2
characteristics.characteristic: title