Comment[GEAAccession]	E-GEAD-887
MAGE-TAB Version	1.1
Investigation Title	Elucidation of disease state by multi-layered omics analysis
Experiment Description	In this project, we investigate the role of omics data, such as genomic, transcriptomic, and other biological information, in the causes and progression of diseases.
Experimental Design	cell type comparison design
Experimental Factor Name	individual
Experimental Factor Type	individual
Person Last Name	Sato	Okada
Person First Name	Go	Yukinori
Person Affiliation	Department of Statistical Genetics, Osaka University Graduate School of Medicine	
Person Roles	submitter	submitter
Public Release Date	2024-12-04
Protocol Name	P-GEAD-3426	P-GEAD-3427	P-GEAD-3428	P-GEAD-3429	P-GEAD-3430	P-GEAD-3431
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	Peripheral blood samples were obtained from patients with NMOSD (n = 25) recruited at Osaka University Hospital. We collected peripheral blood into heparin tubes and isolated PBMCs using Leucosep (Greiner Bio-One) density gradient centrifugation according to the manufacturer's instructions.	We processed single-cell suspension through the 10X Genomics Chromium Controller. Oil droplets of encapsulated single cells and barcoded beads were subsequently reverse transcribed in a Veriti Thermal Cycler, resulting in cDNA tagged with a cell barcode and unique molecular index.	nucleic acid library construction protocol: cDNA was amplified to generate singl-cell libraries according to the manufacturer's protocol. Quantification was made with an Agilent Bioanalyzer High Sensitivity DNA assay (Agilent, High-Sensitivity DNA Kit, 5067-4626). Amplified cDNA was enzymatically fragmented, end-repaired, and poly-A tagged. Cleanup and size selection was performed on amplified cDNA using SPRIselect magnetic beads (Beckman-Coulter, SPRIselect, B23317). Next, Illumina sequencing adapters were ligated to the fragments and cleaned up using SPRIselect magnetic beads. Then, sample indices were selected and amplified, followed by a double-sided size selection using SPRIselect magnetic beads. We assessed the final library quality using an Agilent Bioanalyzer High Sensitivity DNA assay.	nucleic acid sequencing protocol: We sequenced samples on Illumina NovaSeq 6000 as paired-end mode with 90 bp read2 and 20,000 reads depth.	dummy	We processed droplet libraries using Cell Ranger 6.0.0 (10X Genomics). Sequencing reads were aligned with STAR using the GRCh38 human reference genome. We created raw count matrices with the cellranger_count function. We processed them with the remove-background function of CellBender (v0.1.0) to remove cell-free ambient RNAs in the droplets and call cells. We removed cells that had fewer than 1,000 UMIs or greater than 99th percentile of UMIs in each sample, fewer than 200 feature counts or greater than 99th percentile of feature counts in each sample, greater than 12% of reads from mitochondrial genes, or greater than 10% of reads from hemoglobin genes. We further excluded putative doublets using DoubletFinder (v2.0.3) for each sample. We used Seurat (v4.1.0) for data scaling, transformation, clustering, and dimensionality reduction differential expression analysis. We scaled and transformed data using the SCTransform() function, and linear regression was performed to remove unwanted variation due to the percentage of mitochondrial reads. We removed batch effects and integrated scRNA-seq datasets using Scanorama (v1.7.1). We performed a PC analysis based on adjusted gene expression and UMAP using the first 30 PCs.
SDRF File	E-GEAD-887.sdrf.txt
Comment[Number of channel]	single-channel
Comment[DBCLS]	The Data Access Committee of the Database Center for Life Science (DBCLS) approved that this personal data was made published according to the NBDC Guidelines for Human Data Sharing (https://humandbs.dbcls.jp/en/guidelines/data-sharing-guidelines) as the NBDC Research ID hum0197 and the application ID J-DS000220-030.
Comment[Array Design REF]	A-GEAD-11
Comment[AEExperimentType]	transcription profiling by array
Comment[BioProject]	PRJDB19632
Comment[Related study]	NBDC:hum0197
Comment[Last Update Date]	2024-12-04