|
| Status |
Public on Jun 30, 2022 |
| Title |
PBS1 |
| Sample type |
RNA |
| |
|
| Source name |
MACS-sorted GR1+ cells from spleen
|
| Organism |
Mus musculus |
| Characteristics |
lps injection: none
hy209 injection: none
strain: C57BL/6
tissue: Spleen
age: 6~8 week
|
| Treatment protocol |
Spleens were extracted from the mice at 48h after the LPS injection. Gr1+ cells were MACS purified.
|
| Growth protocol |
female C57BL/6 mice were injected with LPS (or PBS) i.p. at time 0, and were injected with HY209 (or PBS) 30 min later.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Gr1+ cells were MACS-soreted from 10-15 spleens of C57BL/6 mice in each group and were pooled together. It was repeated three times to make 3 seperate bilogical samples for microaray. Two to three biological replicates each containing Gr1+ cells (More than 95 % of Gr1+ cells were CD11b+) were amplified using Ambion Illumina TotalPrep RNA Amplification kit. Total RNA was extracted using Trizol (Invitrogen Life Technologies, Carlsbad, USA), purified using RNeasy columns (Qiagen, Valencia, USA) according to the manufacturers’ protocol. After processing with DNase digestion, clean-up procedures, RNA samples were quantified, aliquot and stored at -80°C until use. For quality control, RNA purity and integrity were evaluated by denaturing gel electrophoresis, OD 260/280 ratio, and analyzed on Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA).
|
| Label |
biotin
|
| Label protocol |
Total RNA was amplified and purified using the Ambion Illumina RNA amplification kit (Ambion, Austin, USA) to yield biotinylated cRNA according to the manufacturer’s instructions. Briefly, 550 ng of total RNA was reverse-transcribed to cDNA using a T7 oligo(dT) primer. Second-strand cDNA was synthesized, in vitro transcribed, and labeled with biotin-NTP. After purification, the cRNA was quantified using the ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA).
|
| |
|
| Hybridization protocol |
750 ng of labeled cRNA samples were hybridized to each Mouse Ref8 expression v.2 bead array for 16-18 h at 58°C, according to the manufacturer's instructions (Illumina, Inc., San Diego, USA).
|
| Scan protocol |
Detection of array signal was carried out using Amersham fluorolink streptavidin-Cy3 (GE Healthcare Bio-Sciences, Little Chalfont, UK) following the bead array manual. Arrays were scanned with an Illumina bead array Reader confocal scanner according to the manufacturer's instructions
|
| Description |
Replicate1:negative control for sepsis and drug
|
| Data processing |
The quality of hybridization and overall chip performance were monitored by visual inspection of both internal quality control checks and the raw scanned data. Raw data were extracted using the software provided by the manufacturer (Illumina GenomeStudio v2011.1 (Gene Expression Module v1.9.0)). Probes signal value was transformed by logarithm and normalized by quantile method. Statistical significance of the expression data was determined using LPE test and Fold change in which the null hypothesis was that no difference exists among 2 groups. False discovery rate (FDR) was controlled by adjusting p value using Benjamini-Hochberg algorithm. Gene-Enrichment and Functional Annotation analysis for significant probe list was performed using DAVID (http://david.abcc.ncifcrf.gov/). All data analysis and visualization of differentially expressed genes was conducted using R 2.15.0 (www.r-project.org).
|
| |
|
| Submission date |
Aug 06, 2013 |
| Last update date |
Jun 30, 2022 |
| Contact name |
Seung-yong Seong |
| E-mail(s) |
seongsy@snu.ac.kr
|
| Phone |
+82-2-740-8301
|
| Organization name |
Wide River Institute of Immunology, Seoul National University College of Medicine
|
| Department |
Microbiology and Immunology
|
| Lab |
Hyppo Lab.
|
| Street address |
28 Yongon-dong, Jongno-gu
|
| City |
Seoul |
| ZIP/Postal code |
110-799 |
| Country |
South Korea |
| |
|
| Platform ID |
GPL6885 |
| Series (1) |
| GSE49593 |
mRNA expression profile of regulatory neutrophil induced by sodiumtaurodeoxycholate |
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