Comment[GEAAccession]	E-GEAD-338
MAGE-TAB Version	1.1
Investigation Title	A novel type of antigen presenting cells in mouse
Experiment Description	We analyzed the comprehensive gene expression profiles of novel population of CD135+ monocytes and known populations in mononuclear phagocyte system (MPS) by RNA-Seq to define their hierarchical and relative position in the MPS. Biological replicates were analyzed.
Experimental Design	cell type comparison design
Experimental Factor Name	cell type
Experimental Factor Type	cell type
Person Last Name	Hirai	Naoka	Yuichi	Masakazu
Person First Name	Hideyo	Kamio	Tokuda	Nakano
Person Affiliation	Department of Transfusion Medicine and Cell Therapy, Department of Clinical Laboratory Medicine, Kyoto University Hospital			
Person Roles	submitter			
Public Release Date	2022-05-26
Protocol Name	P-GEAD-337	P-GEAD-338	P-GEAD-339	P-GEAD-340	P-GEAD-341
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol	normalization data transformation protocol
Protocol Description	BM cells obtained from C57BL/6 were stained with multiple monoclonal antibodies and subjected to cell sorting	RNA was extracted with RNeasy micro kit (QIAGEN)	Total RNA was extracted using an RNeasy Micro Kit (Qiagen, Valencia, CA, USA). Quantity and quality of RNA samples from each mouse was confirmed using an Agilent 2200 TapeStation (Agilent). The cDNA libraries for next-generation sequencing (NGS) were constructed using a SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech), a Nextera XT DNA Library Preparation Kit (illumina), and a Nextera XT Index Kit (illumina), according to the manufacturers' instructions.	The quality of NGS libraries was assessed using an Agilent 4200 TapeStation and sequenced on a NovaSeq 6000 System (illumina) using a NovaSeq Xp 4-Lane Kit. Base calls were converted to fastq file format by bcl2fastq2 Conversion Software v2.20 (illumina).	RNA-Seq reads from the fastq files were aligned to Ensembl GRCm38/mm10 genome assembly by using Tophat-2.0.9 with Bowtie2 version 2.1.0 and samtools-0.1.19 after performing the quality control (QC) with FASTX Toolkit 0.0.13, FastQC version 0.11.2, and PRINSEQ lite version 0.20.4. Gene expression analysis was performed on fragments per kilobase million (FPKM) data calculated from the RNA-Seq data using Cufflinks-2.2.1. For the cell clustering, principal component and heatmap analyses were carried out using R version 3.6.0. Finally, 4,891 genes were selected for analyses based on the results from the Cuffdiff option of Cufflinks under the following QC conditions: (i) excluding genes where the \"status\" was not \"OK\", (ii) excluding genes that showed an error value for \"log2(fold_change)\", (iii) including genes that showed \"yes\" for \"significant\", (iv) including genes that resulted in a |log2(fold-change)| >2.0, and (v) excluding genes when more than six samples resulted in FPKM=0. The heatmap images were drawn using the pheatmap R package after normalizing the FPKM values by using zFPKM R/Bioconductor package. The images of RNA-Seq data were drawn using the Integrative Genomics Viewer tool (Robinson et al., 2011).
SDRF File	E-GEAD-338.sdrf.txt
Comment[AEExperimentType]	RNA-seq of coding RNA
Comment[BioProject]	PRJDB9225
Comment[Last Update Date]	2022-05-26