Comment[GEAAccession]	E-GEAD-623
MAGE-TAB Version	1.1
Investigation Title	Transcriptome analysis of fibroblast-dependent alveolar organoids derived from patient-specific iPS cells with SFTPC^Y104H variant and their gene-corrected (monoallelic wild type SFTPC)  ones.
Experiment Description	To investigate genetic variation in alveolar epithelial cells from patients with pulmonary fibrosis, alveolar epithelial cells were generated from patient-derived iPS cells and their gene -corrected patient iPS cells, and transcriptome analysis was performed. *** Related raw data are to be made available through the controlled-access database JGA (study accession JGAS000617, https://humandbs.biosciencedbc.jp/hum0406-v1). ***
Experimental Design	disease state design
Experimental Factor Name	cell_type
Experimental Factor Type	cell_type
Person Last Name	Mikawa	Gotoh
Person First Name	Ryuta	Shimpei
Person Affiliation	Gotoh lab Department of Clinical Application, Center for iPS Cell Research and Application Department of Drug Discovery for Lung Diseases, Graduate of Medicine, Kyoto University	
Person Roles	submitter	submitter
Public Release Date	2023-08-30
Protocol Name	P-GEAD-1864	P-GEAD-1865	P-GEAD-1866	P-GEAD-1867	P-GEAD-1868	P-GEAD-1869
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid labeling protocol	nucleic acid hybridization to array protocol	array scanning and feature extraction protocol	normalization data transformation protocol
Protocol Description	Human fetal lung fibroblasts (17.5 weeks of gestation; DV Biologics #PP002-F-1349) were cultured in DMEM (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich #F7524) and 50 U/mL penicillin-streptomycin (P-S; Thermo Fisher Scientific). Each iPSC line was differentiated stepwise into NKX2-1^+ lung progenitor cells, and CPM^+ cells were isolated using magnetic columns, as described above, on day 14. Seven days later, CPM^high cells were further isolated using fluorescence-activated cell sorting (FACS) with mouse anti-CPM antibody (FUJIFILM Wako Pure Chemical Corporation) as described previously (Korogi et al., 2019). FACS-sorted CPM^high cells (1 x 10^4) were mixed with 5 x 10^5 precultured lung fibroblasts in 200 uL of 50% growth factor reduced Matrigel Matrix (Corning #354230) diluted with DCIK medium (Yamamoto et al., 2017) supplemented with 10 uM Y-27632 (LC Laboratories). Approximately 200 uL of the mixed cells was placed on a 12-well cell culture insert (Corning), and 1 mL of DCIK medium containing 10 uM Y-27632 was added to the lower chamber. In the 24-well format, half the fluid volume was used. FD-AOs were cultured for 14 days, and the medium in the lower chamber was replaced with DCIK medium every two days. This study used a BD FACS Melody Cell Sorter (Becton Dickinson, San Jose, CA, USA) for FACS. Matrigel-embedded cells were carefully dissociated at 37 degrees for 25 minutes via gentle pipetting with 0.1 percent Trypsin-EDTA (Thermo Fisher Scientific). Dissociated cells were stained with Anti-EpCAM/TROP (Bio-Techne #AF960) and CPM (Wako #014-27501) antibodies. After being washed twice with flow cytometry buffer, the cells were stained with the following secondary antibodies. Flow cytometry was performed to separate alveolar epithelial cells (Epi) (EpCAM+CPM+) and fibroblasts (Fib) (EpCAM-CPM-) for the downstream analysis.	The RNeasy Micro Kit was used to extracted the total RNA.	nucleic acid library construction protocol; Sequencinge libraries of the Epi and Fib samplesRNA were prepared using the Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit (Illumina).	nucleic acid sequencing protocol; Sequencing was performed on the Illumina NextSeq2000 with paired-end mode (59bp X 2) reads.	dummy	After trimming adapter sequences and low quality bases from the raw sequenced reads with cutadapt v4.1 (Martin, 2011), the trimmed reads were aligned to human genome (GRCh38) using STAR 2.7.10a (Dobin et al., 2013) with the GENCODE (release 32, GRCh38.p13) (Frankish et al., 2019). The raw counts were calculated using htseq-count ver. 2.02 (Anders et al., 2015) with the GENCODE gtf file.
SDRF File	E-GEAD-623.sdrf.txt
Comment[Number of channel]	single-channel
Comment[Array Design REF]	A-GEAD-11
Comment[AEExperimentType]	transcription profiling by array
Comment[NBDC]	The Data Access Committee of the National Bioscience Database Center (NBDC) approved that this personal data was made published according to the NBDC Guidelines for Human Data Sharing (https://humandbs.biosciencedbc.jp/en/guidelines/data-sharing-guidelines) as the NBDC Research ID hum0406 and the application ID J-DS000923-001.
Comment[BioProject]	PRJDB16121
Comment[Related study]	JGA:JGAS000617	NBDC:hum0406
Comment[Last Update Date]	2023-08-30