|
| Status |
Public on May 02, 2017 |
| Title |
day 3 definitive endoderm 2 |
| Sample type |
RNA |
| |
|
| Source name |
iPSC-derived CXCR4+ definitive endoderm
|
| Organism |
Homo sapiens |
| Characteristics |
group: definitive endoderm
timepoint: day 3
|
| Treatment protocol |
Not applicable
|
| Growth protocol |
iPSCs were transitioned to feeder-free conditions on Matrigel (Corning) in mTeSR1 (Stem Cell Technologies) and passaged with Gentle Cell Dissociation Reagent (Stem Cell Technologies). For the lung differentiation we first induced definitive endoderm using STEMDiff definitive endoderm kit (STEMCELL Technologies) according to the accompanying protocol. After approximately 72 cells were plated in small clumps at approximately 150-200,000 cells/cm2 on Matrigel-coated plates in complete serum-free differentiation media (CSFDM) supplemented with 2μM Dorsomorphin (Stemgent), 10μM SB431542 (Tocris) for 72 hours. 10μM Y-27632 (Tocris) was added for the first 24 hours. The differentiation media was changed on day 6 to “CFKBRa”; CSFDM supplemented with 3μM CHIR99021 (Tocris), 10ng/ml rhFGF10, 10ng/ml rhKGF, 10ng/ml rhBMP4 (all from R&D Systems), 50-100nM Retinoic acid (Sigma). On day 15 cells were dissociated by incubating in 0.05% trypsin (ThermoFisher Scientific) at 37°C for 2-4 minutes, aspirating trypsin, washing once with DMEM (ThermoFisher Scientific)+10% FBS (ThermoFisher Scientific), resuspending as small clumps in CSFDM supplemented with 3uM CHIR99021, 10ng/ml rhFGF10 and 10ng/ml rhKGF (“CFK” media) and plated on freshly-coated Matrigel (Corning 354277) plates. 10μM Y-27632 was added to “CFK” media for the first 24 hours. On day 22 media was changed to “CFK+DCI”: “CFK” media plus 50nM dexamethasone (Sigma), 0.1mM 8-Bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) sodium salt (Sigma) and 0.1mM 3-Isobutyl-1-methylxanthine (IBMX) (Sigma). Neuroectodermal NKX2-1GFP+ cells were generated using STEMDdiff Neural Induction Medium (STEMCELL Technologies) according to the manufacturer’s protocol. On day 6 of differentiation, 2μM of purmorphamine (Stemgent) was added to the base media. NKX2-1+ cells were sorted on Day 12.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted by first lysing cells in Qiazol (Qiagen) and subsequently using RNeasy Plus Minikit (Qiagen) according to the manufacturer’s protocol and without DNase treatment.
|
| Label |
biotin
|
| Label protocol |
Biotin labeling was performed using the Ambion WT Expression Kit (Life Technologies, Grand Island, NY) according to the manufacturer's protocol, followed by the GeneChip WT Terminal Labeling and Controls Kit (Affymetrix, Santa Clara, CA).
|
| |
|
| Hybridization protocol |
The labeled, fragmented DNA was hybridized to the Human Gene 2.0 ST Array (Affymetrix, Santa Clara, CA) for 18 hours in a GeneChip Hybridization oven 640 at 45oC with rotation (60 rpm). The hybridized samples were washed and stained using an Affymetrix fluidics station 450.
|
| Scan protocol |
After staining, microarrays were immediately scanned using an Affymetrix GeneArray Scanner 3000 7G Plus (Affymetrix, Santa Clara, CA).
|
| Description |
5
Gene expression data from iPSC-derived CXCR4+ definitive endoderm
|
| Data processing |
Raw Affymetrix CEL files were normalized to produce Entrez Gene-identifier-specific expression values using the implementation of the Robust Multiarray Average (RMA) in the affy package in the Bioconductor software suite (version 2.12), using R version 2.15.1 and the Brainarray hugene20sthsentrezg R package (version 17.0.0) (http://mbni.org/customcdf/17.0.0/entrezg.download/hugene20st_Hs_ENTREZG_17.0.0.zip).
|
| |
|
| Submission date |
Jun 13, 2016 |
| Last update date |
Jun 25, 2019 |
| Contact name |
Boston University Microarray and Sequencing Resource |
| E-mail(s) |
msrdata@bu.edu
|
| Organization name |
Boston University
|
| Department |
Microarray and Sequencing Resource
|
| Street address |
72 East Concord Street, E631
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02118 |
| Country |
USA |
| |
|
| Platform ID |
GPL17930 |
| Series (1) |
| GSE83310 |
Global gene expression kinetics of early human lung development modeled by directed differentiation of human PSCs using an NKX2-1GFP iPSC reporter |
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