Comment[GEAAccession]	E-GEAD-674
MAGE-TAB Version	1.1
Investigation Title	CD4 T cell heterogeneity and clonal expansion depend on gestational age and is altered in preeclampsia by single cell RNA sequence
Experiment Description	We isolated decidual CD4 T cells from healthy early gestation, healthy late gestation, early onset PE with a diagnosis before 34 weeks and delivery between 34 - 37 weeks of gestation. CD4 T cells from peripheral blood mononuclear cells (PBMC) of one case of healthy late gestation was included. We conducted single-cell RNA sequences and single cell T cell receptor analysis of them to study human CD4 T cell at feto-maternal interface from healthy early gestation, healthy late gestation and preeclampsia. hST02_TsudaTar2, hST03_TsudaTar3 and hST04_TsudaTar3 are healthy late gestation decidua. hST03_TsudaTar2 is a normal term delivery PBMC. hST04_TsudaTar2,hST05_TsudaTar2,hST01_TsudaTar3 and hST02_TsudaTar3  are healthy early gestation decidua. hST06_TsudaTar2, hST05_TsudaTar3 and hST06_TsudaTar3 are preeclampsia decidua.
Experimental Design	observational design
Experimental Factor Name	disease
Experimental Factor Type	disease
Person Last Name	Tsuda
Person First Name	Sayaka
Person Affiliation	University of Toyama
Person Roles	submitter
Public Release Date	2024-04-26
Protocol Name	P-GEAD-2134	P-GEAD-2135	P-GEAD-2136	P-GEAD-2137	P-GEAD-2138	P-GEAD-2139
Protocol Type	sample collection protocol	nucleic acid extraction protocol	nucleic acid library construction protocol	nucleic acid sequencing protocol	normalization data transformation protocol	high throughput sequence alignment protocol
Protocol Description	Decidua was rinsed with PBS until the blood was removed, minced with a pair of scissors to produce 1 to 2mm pieces, and filtered through 41um nylon net filter. The filtered cell suspension was the layered Ficoll Hypaque and centrifuged for 1500rpm, 30min with brakes off. Immune cells at the interface of gradients were collected and cryopreserved. Peripheral blood mononuclear cells are also isolated by density gradient centrifuging, the same as decidual immune cells. CD4 T cells were sorted from cryopreserved mononuclear cells by cell sorter.	Isolated CD4 T cells were loaded onto BD Rhapsody cartrige. Cell load, cell lysis, mRNA trap were conducted according to the manufacture's instruction.	scTCR and Targeted RNAseq libraries of CD4 T cells were constructed by using a BD Rhapsody system with BD Rhapsody Targeted mRNA and AbSeq Reagent kit by BD Bioscience, and BD Rhapsody T Cell Expression Panel Hs by BD Bioscience with human IL-10 and NRP1 primers supplement by BD Bioscience according to the instruction of the manufacture, except cDNA denaturation was performed by using 0.1M NaOH for 5 min at room temperature.	Sequencing was performed by using an Illumina Novaseq 6000 and Novaseq S4 flowcells (200 cycles kit, read 1 67bp, read 2 155bp) by ImmunoGeneTeqs, Inc. (Chiba, Japan).	Adapter trimming and quality filtering were performed. Base composition of sequencing data was analyzed by FastQC v0.11.9. Pair end Fastq files of samples were processed. R1 is cell barcode reads, R2 is RNA reads. Filtered cell barcode reads were annotated by Python script provided by BD with minor modification for compatible to Python3.8. Associated cDNA reads were mapped to reference RNA. Cell barcode information of each read was added to the bowtie2 mapped BAM files and read counts of each gene in each cell barcode were counted. Cells with the total read count was over inflection point were regarded as valid cells. For assignment of each tag to each cell barcode, read counts of each tag in each valid cell barcode, which defined by the cDNA matrix, were extracted from tag and cell barcode expression matrix. Unassigned cell barcodes were labeled as not-detected cells. Then, the sum of the total read counts of each tag were normalized to 10M reads, and log2 fold change between first most counted tags and second most counted tags within each cell barcode. Doublet cells which are double positive cells of any pair of Tags were identified by flowDensity package. Log2 fold change between first and second most counted tags less than 0.2415 were identified as doublets. Finally, remaining cell barcodes were assigned to the first most counted tags. Each tag expression in each cell barcode were log2 transformed, z scaled by each cell barcode.  TCR data matrixes which contains VDJ read counts, nucleotide sequences and amino acid sequences of CDR3, and gene usages were provided by ImmunoGeneTeqs, Inc., Chiba, Japan. Cells with paired alpha/beta chain were used for TCR analysis. Distribution-based error correction which is included BD Rhapsody targeted scRNA seq workflow in order to reduce background read counts of each gene that were possibly derived from RNA diffusion during cell lysis step within BD Rhapsody cartridge and reverse transcription was conducted by ImmunoGeneTeqs, Inc., Chiba, Japan.	GRCh38 release-101
SDRF File	E-GEAD-674.sdrf.txt
Comment[AEExperimentType]	RNA-seq of coding RNA from single cells
Comment[BioProject]	PRJDB17285
Comment[Last Update Date]	2024-04-26