title: MICHIGAN ORGANOID @10D_1
organism: Homo sapiens
description: Protocols: Differentiation of hESCs and organoids was carried out as previously published, except where noted in the text (Finkbeiner et al., 2015b; McCracken et al., 2011). In brief, endoderm was generated by adding ActivinA (100ng/mL) for 3 consecutive days in RPMI supplemented with 0%, 0.2% and 2.0% HyClone FBS. FGF4 (500ng/mL) plus CHIR99021 (2uM) were then added to endoderm cultures, and media was replaced daily for 10 days. Spheroids were collected on d5, d7 and d10, and were embedded in matrigel, and overlayed with growth medium as previously described (Finkbeiner et al., 2015b) containing RSPO2 conditioned medium (Bell et al., 2008), NOGGIN conditioned medium (Heijmans et al., 2013) and EGF. RNA was isolated directly from tissue culture plates using RNeasy Mini Kit (Qiagen). Media was aspirated and for organoids Matrigel was allowed to dissolve for 1 hour at room 4 degrees. Lysis buffer added directly onto plates using RLT buffer (Qiagen) and RNA isolated as per protocol. RNA concentration and purity were assessed using Nanodrop spectrophotometer and bioanalyzed using Agilent RNA 6000 Nano Kit (260/280 > 1.7 and 260/230 >1.7, RIN>8). We used 10ng of RNA to generate libraries (TrueSeq Kit, Illumina). A small aliquot of libraries were bioanalyzed and run on MiSeq to allow for normalization of reads.
attributes.tag: ENA first public
attributes.value: 2016-01-02
attributes.tag: ENA last update
attributes.value: 2015-12-23
attributes.tag: External Id
attributes.value: SAMEA3719373
attributes.tag: INSDC center alias
attributes.value: UCSF Department of Pediatrics, Division of Pediatric Gastroenterology
attributes.tag: INSDC center name
attributes.value: UCSF Department of Pediatrics, Division of Pediatric Gastroenterology
attributes.tag: INSDC first public
attributes.value: 2016-01-02T17:01:19Z
attributes.tag: INSDC last update
attributes.value: 2015-12-23T15:38:04Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-4168:MICHIGAN ORGANOID @10D_1
attributes.tag: age
attributes.value: 10
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: embryonic stem cells derived human intestinal organoids
attributes.tag: common name
attributes.value: human
attributes.tag: growth condition
attributes.value: CHIR99021/FGF4-enriched media
attributes.tag: sample name
attributes.value: E-MTAB-4168:MICHIGAN ORGANOID @10D_1