|
| Status |
Public on Aug 25, 2020 |
| Title |
CD11b Ly6C– cult 2 |
| Sample type |
RNA |
| |
|
| Source name |
Mus musculus
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
Sex: male
cell type: Ly-6C+ CD11b+ cDCs
|
| Treatment protocol |
C57BL/6 mice were inhaled house dust extract (10 micrograms) together with ovalbumin (100 micrograms). Sixteen hours later, lungs were excised, and Ly-6C+CD11b+, Ly-6C– CD11b+, and CD103+ cDCs (CD11c+ I-A+ CD45+ CD88– Siglec-F– F4/80– Live/Dead–) and monocytes (CD45+ F4/80+ CD88– Siglec-F– I-A– Live/Dead–) were purified by flow cytometric sorting. Purified lung cDCs and monocytes (2×105 cells/200 μL/well) were cultured for 2 days in complete RPMI1640 medium containing 10% fetal bovine serum. Total RNA (50 ng of each ) was isolated from freshly isolated or cultured cells, and mRNA expression was examined using the NanoString platform utilizing the Mouse Myeloid Innate Immunity Panel v2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was isolated from cells using Trizol, Dnase treated with Dnase I and further purified using Qiagen Rneasy mini kits. RNA concentration and purity was determined using a Qubit and Nanodrop.
|
| Label |
na
|
| Label protocol |
n/a
|
| |
|
| Hybridization protocol |
Reporter and Capture probes were added according to the manufacturer’s directions (nanostring.com) to 50 ng of total RNA in a final volume of 15ul followed by hybridization for at least 14 hours at 65oC
|
| Scan protocol |
After sample processing according to manufacturer’s instructions on the nCounter Prep Station Model 5S, sample cartridges were scanned on an nCounter Digital Analyzer Model 5S, using 555 Fields of Vision (FOV) and the .rlf file 'Ns_Mm_Myeloid_v2'.
|
| Data processing |
Data were adjusted utilizing the manufacturer’s positive and negative experimental control probes, as well as 2 HK genes (Sap130 and Sdha). All samples passed nSolver’s initial QA/QC controls. Compiled raw and data adjusted with positive/negative controls and the HK genes were exported as .csv files. nSolver adjusted data were then imported into Partek_v7.0 for further analysis
|
| |
|
| Submission date |
Aug 24, 2020 |
| Last update date |
Aug 25, 2020 |
| Contact name |
Rick David Fannin |
| E-mail(s) |
fannin@niehs.nih.gov
|
| Phone |
9842874069
|
| Organization name |
Mr.
|
| Department |
NIEHS/NIH
|
| Lab |
Genomics Core
|
| Street address |
111 Tw Alexander Dr, MD D204
|
| City |
RTP |
| State/province |
NC |
| ZIP/Postal code |
27709 |
| Country |
USA |
| |
|
| Platform ID |
GPL25266 |
| Series (1) |
| GSE156763 |
Gene expression of mouse lung CD11b+ dendritic cells before and after ex vivo culture |
|
