|
| Status |
Public on Jul 24, 2020 |
| Title |
Subject 31_non-SS_Batch 3 |
| Sample type |
RNA |
| |
|
| Source name |
non-SS_Batch 3_Minor salivary gland memory CD4 T cells
|
| Organism |
Homo sapiens |
| Characteristics |
subject id: Subject 31
aecg disease classification: non-SS
age: 37
Sex: M
race: More Than One
batch: Batch 3
cell type: Minor salivary gland memory CD4 T cells
|
| Treatment protocol |
SG biopsy tissue (4-6 minor salivary glands/subject) was minced, digested with 750 U/mL Collagenase I (Sigma), 500 U/mL Hyaluronidase IV (Sigma) and 0.1 mg/mL DNAse I (Roche) in RPMI/10mM Hepes/5% fetal calf serum (FCS) for 1 hour at 37°C, then dissociated with program “B” on a gentleMACS instrument (Miltenyi Biotec) or processed as described in PMID27358913. Cells were stained with monoclonal antibodies (CD3-PE, CD4-PECy5, CD8-Alexa 488 and CD45RA-V450, Becton Dickinson, Franklin Lakes, NJ, USA). CD3+CD4+CD45RA- cells excluding propidium iodide were first bulk sorted at high purity using doublet discrimination on a FACSAria (Becton Dickinson). Then 200 cells in ~1 µL were sorted into 6.7 µL SuperAMP buffer (Miltenyi Biotec, Auburn, CA, USA) using a MoFlo-XDP (Beckman-Coulter Indianapolis, IN, USA) and stored at -80 degrees C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were shipped on dry ice to Miltenyi Biotec where mRNA isolated with paramagnetic oligo(dT) microbeads underwent proprietary bead-bound cDNA synthesis, 3′-end tailing and labeling, followed by single-primer amplification of cDNA and cDNA purification.
|
| Label |
Cy3
|
| Label protocol |
250 ng of each cDNA sample was used as template for Cy3 labeling which was performed according to Miltenyi Biotec's undisclosed protocol.
|
| |
|
| Hybridization protocol |
The Cy3-labeled cDNAs were hybridized overnight (17 hours, 65 degrees C) to an Agilent Whole Human Genome Oligo Microarray 8x60K using Agilent's recommended hybridization chamber and oven. The microarrays were washed once with Agilent Gene Expression Buffer 1 for 1 min at room temperature followed by a second wash with preheated (37 degrees C) Agilent Gene Expression Wash Buffer 2 for 1 min.
|
| Scan protocol |
Fluorescence signals of the hybridized Agilent Microarrays were detected using Agilent's Microarray Scanner System (Agilent Technologies).
|
| Data processing |
The Agilent Feature Extraction Software (FES) was used to read out the microarray image files. Amplified cDNA samples from 17 pSS and 15 nSS subjects were hybridized to Agilent Whole Human Genome 8x60K microarrays in three batches. All data were pooled to assess potential batch effects by principal components analysis, and gene expression data were quality checked using the arrayQualityMetrics R package. Batch effects were equalized via ComBat analysis (sva R package v 3.8.0; manual specification of batches). Low-variability genes (defined as <50% of the overall variability distribution) were filtered using the function varFilter in R to reduce the false positive rate. The data were quantile normalized, and differentially expressed genes were detected using the limma R package v3.3. P-values were corrected for multiple testing using the Benjamin-Hochberg procedure. The significance threshold was set at a false discovery rate (FDR) of 0.1 resulting in adjusted p-values less than 0.1. Genes with log fold-change values of at least 1.5 were included in the list of differentially expressed genes (DEG). Data from multiple probes for the same gene were collapsed to one by maximum expression level.
|
| |
|
| Submission date |
Jan 06, 2020 |
| Last update date |
Jul 25, 2020 |
| Contact name |
A. Darise Farris |
| E-mail(s) |
FARRISD@OMRF.ORG
|
| Phone |
405-271-7389
|
| Organization name |
Oklahoma Medical Research Foundation
|
| Department |
Arthritis & Clinical Immunology
|
| Street address |
825 NE 13th Street, MS 24
|
| City |
Oklahoma City |
| State/province |
OK |
| ZIP/Postal code |
73104 |
| Country |
USA |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE143153 |
Microarray analysis of salivary gland CD4+ T cells |
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