|
| Status |
Public on Jan 04, 2022 |
| Title |
Monocytes+LPS (0.5ug/ml)_1 |
| Sample type |
RNA |
| |
|
| Source name |
in vitro cultured Purified Monocytes
|
| Organism |
Mus musculus |
| Characteristics |
experimental group: Monocytes +LPS (0.5ug/ml)
treatment: LPS (0.5ug/ml)
Sex: Female
age: 8-12weeks
cell type: in vitro cultured Purified Monocytes
|
| Treatment protocol |
Mice colonies were maintained at Baylor college of Medicne vivarium under the standard protocol
|
| Growth protocol |
C57B/6 Mice were purchased from Jackson lab AND Pinkie mice were inhouse breed and maintained at BCM animal facilities until use
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from cultured cells
Whole cornea was surgically removed or the in vitro cultured Immune cells were spun down and the RNA was extracted using the standard protocol provided by the manufacturer, we use the Qiagen RNA preparation kit
|
| Label |
n/a
|
| Label protocol |
The samples were labeled according to the condition
|
| |
|
| Hybridization protocol |
One hundred nanograms of total RNA were hybridized with the Nanostring Technologies nCounter Mouse Immunology Panel and Nanostring Technologies nCounter Gene Expression Mouse Myeloid Innate Immunity Panel codeset (NS_MM_Myeloid_V2.0+Pls) containing 770 Unique pairs of 35-50bp reporter probes, 12 custom probe pairs and biotin-labeled capture probes, including internal refereance controls. overnight hybridization occured for 20hours at 65C.
|
| Scan protocol |
The cartridge containing immobilized sample was transferred to the nCounter Digital Analyzer (Software v3.0.1.4) and scanned at 555 field of view (FOV).
|
| Description |
Mono_LPS1
|
| Data processing |
Data was processed using ROSALIND (https://app.rosalind.bio)
Data was analyzed by ROSALIND® (https://rosalind.bio/), with a HyperScale architecture developed by ROSALIND, Inc. (San Diego, CA). Read Distribution percentages, violin plots, identity heatmaps, and sample MDS plots were generated as part of the QC step. Normalization, fold changes and p-values were calculated using criteria provided by Nanostring. ROSALIND® follows the nCounter® Advanced Analysis protocol of dividing counts within a lane by the geometric mean of the normalizer probes from the same lane. Housekeeping probes to be used for normalization are selected based on the geNorm algorithm as implemented in the NormqPCR R library1. Abundance of various cell populations is calculated on ROSALIND using the Nanostring Cell Type Profiling Module. ROSALIND performs a filtering of Cell Type Profiling results to include results that have scores with a p-Value greater than or equal to 0.05. Fold changes and pValues are calculated using the fast method as described in the nCounter® Advanced Analysis 2.0 User Manual. P-value adjustment is performed using the Benjamini-Hochberg method of estimating false discovery rates (FDR). Clustering of genes for the final heatmap of differentially expressed genes was done using the PAM (Partitioning Around Medoids) method using the fpc R library2 that takes into consideration the direction and type of all signals on a pathway, the position, role and type of every gene, etc. Hypergeometric distribution was used to analyze the enrichment of pathways, gene ontology, domain structure, and other ontologies. The topGO R library3, was used to determine local similarities and dependencies between GO terms in order to perform Elim pruning correction. Several database sources were referenced for enrichment analysis, including Interpro4, NCBI5, MSigDB6,7, REACTOME8, WikiPathways9. Enrichment was calculated relative to a set of background genes relevant for the experiment.
|
| |
|
| Submission date |
Jan 03, 2022 |
| Last update date |
Jan 04, 2022 |
| Contact name |
JEHAN ALAM |
| E-mail(s) |
[email protected]
|
| Phone |
(402) 686-7021
|
| Organization name |
Baylor College of Medicine
|
| Street address |
Baylor One Plaza
|
| City |
Houston |
| State/province |
TEXAS |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL31165 |
| Series (2) |
| GSE192960 |
Reduced RXRa signaling increases dry eye disease inducing gdT17 cells in the conjunctiva [Nanostring] |
| GSE195962 |
Reduced RXRa signaling increases dry eye disease inducing gdT17 cells in the conjunctiva |
|
