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Sample GSM4718733

Query DataSets for GSM4718733

Status

Public on Aug 11, 2020

Title

hPSC_Lung organoid_SARS-CoV-2_imatinib_3 [LungOrganoid-10uM-Imatinib-3]

Sample type

SRA

Source name

LungOrganoid-10uM-Imatinib-3

Organism

Homo sapiens

Characteristics

tissue/cell type: hPSC_Lung organoid
treatment: SARS-CoV-2+imatinib

Treatment protocol

hPSC-LOs were infected with SARS-CoV-2 at the indicated MOI and incubated for 24 hrs at 37°C. hPSC_LOs were treated with DMSO or 10 uM imatinib and infected with SARS-CoV-2 at indicated MOI.

Growth protocol

Protocols for maintenance of hPSCs and generation of lung organoids were slightly modified from previous studies. The hESC line RUES2 was cultured on irradiated mouse embryonic fibroblasts (Global Stem, cat. no. GSC-6001G) at a density of 20,000- 25,000 cells/cm2 in a medium of DMEM/F12, 20% knockout serum replacement (Life Technologies), 0.1 mM β-mercaptoethanol (Sigma Aldrich) and 20 ng/ml bFGF (R&D Systems), and medium was changed daily. hESC cultures were maintained in an undifferentiated state at 37 °C in a 5% CO2/air environment until stem cells reached about 90% confluence. hESC differentiation into endoderm was performed in serum-free differentiation (SFD) medium of DMEM/F12 (3:1) (Life Technologies) supplemented with N2 (Life Technologies), B27, 50 μg/ml ascorbic acid, 2 mM Glutamax, 0.4 μM monothioglycerol, 0.05% BSA at 37 °C in a 5% CO2/5% O2/95% N2 environment. hESCs were treated with Accutase and plated onto low attachment 6-well plates (Corning Incorporated, Tewksbury MA), resuspended in endoderm induction medium containing 10 μM Y-27632, 0.5 ng/ml human BMP4, 2.5 ng/ml human bFGF, 100 ng/ml human Activin A, for 72-76 hours dependent on the formation rates of endoderm cells. On day 3 or 3.5, the endoderm bodies were dissociated into single cells using 0.05% Trypsin/0.02% EDTA and plated onto fibronectin-coated, 24-well tissue culture plates (~100,000–150,000 cells/well). For induction of anterior foregut endoderm, the endoderm cells were cultured in SFD medium supplemented with 1.5 μM dorsomorphin dihydrochloride (R&D Systems) and 10 μM SB431542 (R&D Systems) for 36 hrs, and then switched to 36 hrs of 10 μM SB431542 and 1 μM IWP2 (R&D Systems) treatment. For induction of early stage lung progenitor cells (day 6–15), the resulting anterior foregut endoderm was treated with 3 μM CHIR99021, 10 ng/ml human FGF10, 10 ng/ml human KGF, 10 ng/ml human BMP-4 and 50-60 nM all-trans retinoic acid (ATRA), in SFD medium for 8–10 d. The day 10–15 cultures were maintained in a 5% CO2/air environment. On days 15 and 16, the lung field progenitor cells were replated after one minute trypsinization onto fibronectin-coated plates, in the presence of SFD containing either a combination of five factors (3 μM CHIR99021, 10 ng/ml human FGF10, 10 ng/ml human FGF7, 10 ng/ml human BMP4, and 50 nM ATRA). Day 16–25 cultures of late stage lung progenitor cells were maintained in SFD media containing 3 μM CHIR99021, 10 ng/ml human FGF10, 10 ng/ml human KGF, in a 5% CO2/air environment. For differentiation of mature lung cells (day 25 to 55) in 3D culture, cultures were re-plated and embedded in 90% Matrigel after brief trypsinization in SFD media containing maturation components containing 3 μM CHIR99021, 10 ng/ml human FGF-10; 10 ng/ml human KGF, and DCI (50 nM Dexamethasone, 0.1 mM 8-bromo-cAMP (Sigma Aldrich ) and 0.1 mM IBMX (3,7-dihydro-1-methyl-3-(2-methylpropyl)-1H-purine-2,6-dione) (Sigma Aldrich).

Extracted molecule

total RNA

Extraction protocol

At 24 hpi, hPSC-LOs were washed three times in PBS and lysed in TRIzol for RNA analysis.
Libraries were prepared using Illumina TruSeq stranded mRNA Sample Preparation kit.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Illumina bcl2fastq2 v2.20 was used for basecalling.
Sequenced reads were trimmed for low-quality bases and for adapter sequences using cutadapt; then mapped to the human hg19 reference genome plus SARS-CoV-2 genome using STAR v2.5.2b.
Transcript abundances was estimated in Reads Per Kilobase of exon per Million mapped reads (RPKM) by using Cufflinks v2.1.1 package.
Genome_build: hg19
Supplementary_files_format_and_content: excel files storing RPKM per gene per sample

Submission date

Aug 10, 2020

Last update date

Aug 11, 2020

Contact name

Shuibing Chen

E-mail(s)

shuibing.chen@gmail.com

Phone

2127465431

Organization name

Weill Cornell Medical College