title: PB_RNA
organism: Homo sapiens
description: polyA+ RNA 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. RNA was extracted usign TRIzol. Total RNA quality was confirmed using Bioanalyzer 2100. All analyzed samples had RIN values greater than 9. RNA-seq for in vivo and in vitro MPCs was generated from DNase I-treated PolyA+ RNA samples from a CS17 pancreatic bud or from a pool of 3 in vitro pancreatic MPC differentiation experiments. Libraries were contructed using Illumina standard recommended procedures.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2015-04-06T17:04:46Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T21:57:28Z
attributes.tag: External Id
attributes.value: SAMEA3121277
attributes.tag: INSDC center name
attributes.value: IDIBAPS and IFIBYNE-CONICET
attributes.tag: INSDC first public
attributes.value: 2015-04-06T17:04:46Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T21:57:28Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-3061:PB_RNA
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: Carnegie Stage 17
attributes.tag: organism part
attributes.value: pancreatic bud
attributes.tag: sample name
attributes.value: E-MTAB-3061:PB_RNA
attributes.tag: scientific_name
attributes.value: Homo sapiens