Comment[GEAAccession]	E-GEAD-811
MAGE-TAB Version	1.1
Investigation Title	RNA sequencing and analysis of human eosinophils stimulated with all-trans retinoic acid and IL-5
Experiment Description	Activated eosinophils is a major cell type to be involved in allergic diseases. IL-5 primarily activates eosinophils and prolongs their survival. In contrast, all-trans retinoic acid has regulatory effects in the immune system. The aim of this study is to clarify the role of all-trans retinoic acid in the regulation of human eosinophils.
Experimental Design	cellular modification design	stimulus or stress design	replicate design
Experimental Factor Name	treatment
Experimental Factor Type	treatment
Person Last Name	Miyata
Person First Name	Jun
Person Affiliation	Division of Pulmonary Medicine, Department of Medicine, Keio University
Person Roles	submitter
Public Release Date	2025-06-23
Protocol Name	P-GEAD-3016	P-GEAD-3017	P-GEAD-3018	P-GEAD-3019	P-GEAD-3020
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol	normalization data transformation protocol
Protocol Description	Human eosinophils were isolated from peripheral blood. Erythrocyte was removed using 0.9% dextran solution. The fraction of granulocyte including eosinophils and neutrophils were obtained using Percoll reagent. Hemolytic method was performed and recovered using MQ water and its equal amount of 1.8% NaCl solution. We used a negative selection method with biotin-conjugated antibodies against other cell surface markers (CD2, CD14, CD16, CD19, CD56, CD123, and CD235a) and anti-biotin microbeads to isolate eosinophils from the peripheral blood of healthy volunteers according to the protocols of the manufacturers (Miltenyi Biotec, Bergisch Gladbach, Germany). Eosinophils were stimulated with ATRA or IL-5 for 72 h. After stimulation, eosinophils were lysed with 0.5 mL of TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA) and stored until use.	Total RNAs were isolated from the lysates according to the manufacturer's protocol. Breifly, Lysates were incubated for 5 minutes to permit complete dissociation of the nucleoproteins complex and 0.2 mL of chloroform per 1 mL of TRIzol Reagent used for lysis was added. After incubation for 2 to 3 minutes, the sample was centrifuged and the mixture separates into a lower red phenol-chloroform, and interphase, and a colorless upper aqueous phase. The aqueous phase containing the RNA was transferred to a new tube. After this procedure, the RNA was precipitated using isopropanol, washed with 75% ethanol, and solubilized in RNase-free water.	The concentration and the quality of the RNA were verified using a Qubit fluorometer (Thermo) and Agilent 2100 bioanalyzer, respectively. Purified total RNA was used for RNA library preparation, according to the instructions of the Quant Seq 3'mRNA-seq library preparation kit FWD of Illumina (Lexogen, Vienna, Austria). Briefly, cDNA was synthesized using oligo (dT) primers from 25 ng of Total RNA. Template RNA was removed from the sample. Double strand cDNA was synthesized using random primers. After this procedure, PCR was performed for 16 cycles using adapters for Illumina sequencers. Second strand synthesis was followed by a magnetic bead-based purification step.	After the step of library amplification, the RNA libraries were sequenced on an Illumina NextSeq 500 system with 75-nt-long reads. NextSeq 500/550 High Output Kit v2.5 was used to repeat 75 cycles. Prior to mapping, the adapter sequences were removed from the raw reads, and base trimming was performed from the 3' end of each read to remove bases with quality below Q10 up to a minimum length of 25 bp. Reads shorter than 25 bp were removed prior to further analysis. Each read was mapped to the human genome hg38 using Strand NGS (v4, Strand Life Sciences) using default settings.	Normalization of DESeq was performed using default settings of the tools DESeq (Differential Expression analysis for Sequence data). The raw read counts for each gene are adjusted based on the total number of reads in each sample. This step accounts for differences in sequencing depth between samples. DESeq calculates a normalization factor for each sample to scale the library sizes. These factors are determined based on the median of the ratios of observed counts to expected counts across all genes in each sample. Optionally, DESeq can apply a variance stabilizing transformation (VST) or a regularized logarithm (rlog) transformation to the normalized counts. This transformation stabilizes the variance of the counts, making them more suitable for downstream statistical analysis.
SDRF File	E-GEAD-811.sdrf.txt
Comment[AEExperimentType]	RNA-seq of coding RNA
Comment[BioProject]	PRJDB18171
Comment[Last Update Date]	2025-06-23