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
24 Gene expression data from iPSC-derived forebrain-like neural NKX2-1GFP+ cells
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