|
| Status |
Public on Mar 21, 2018 |
| Title |
Control LPS-stimulated RAW264.7 cells Replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Control LPS-stimulated RAW264.7 cells
|
| Organism |
Mus musculus |
| Characteristics |
cell line: RAW264.7
cell type: Macrophage-like
molecule: Sub-polysomal RNA
treatment: DMSO
|
| Treatment protocol |
Cells were incubated with DMSO carrier or 125ng/ml mycolactone for 1hr then stimulated with 100ng/ml LPS for 4hr
|
| Growth protocol |
RAW264.7 cells were grown in high glucose DMEM supplemented with 10% fetal bovine serum
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells treated with 10µg/ml cyclohexhamide for 10min and lysed in 15mM Tris pH7.4, 300mM NaCl, 15mM MgCl2, 100µg/ml cyclohexamide, 1mg/ml heparin. Polysomal and sub-polysomal RNA prepared by 10-60% sucrose gradient centrifugation and purified by LiCl precipitation
|
| Label |
Cy3
|
| Label protocol |
0.15µg polysomal or subpolysomal RNA was spiked with Agilent RNA Spike-in Kit and labelled using Agilent Low input Quick Amp 2-colour labelling kit according to manufacturer's instructions
|
| |
|
| Channel 2 |
| Source name |
Control LPS-stimulated RAW264.7 cells
|
| Organism |
Mus musculus |
| Characteristics |
cell line: RAW264.7
cell type: Macrophage-like
molecule: Polysomal RNA
treatment: DMSO
|
| Treatment protocol |
Cells were incubated with DMSO carrier or 125ng/ml mycolactone for 1hr then stimulated with 100ng/ml LPS for 4hr
|
| Growth protocol |
RAW264.7 cells were grown in high glucose DMEM supplemented with 10% fetal bovine serum
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells treated with 10µg/ml cyclohexhamide for 10min and lysed in 15mM Tris pH7.4, 300mM NaCl, 15mM MgCl2, 100µg/ml cyclohexamide, 1mg/ml heparin. Polysomal and sub-polysomal RNA prepared by 10-60% sucrose gradient centrifugation and purified by LiCl precipitation
|
| Label |
Cy5
|
| Label protocol |
0.15µg polysomal or subpolysomal RNA was spiked with Agilent RNA Spike-in Kit and labelled using Agilent Low input Quick Amp 2-colour labelling kit according to manufacturer's instructions
|
| |
|
| |
| Hybridization protocol |
Pooled labelled cRNAs were fragemented at 60⁰C for 30min then applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After overnight hybridization at 65⁰C in Agilent Hi-RPM Hybridisation buffer, slides were washed according to manufacturer's instructions.
|
| Scan protocol |
Scanned on an Agilent G2505C scanner
Images were quantified using Agilent Feature Extraction Software (version A.8.5.1.1).
|
| Description |
Biological replicate 2 of 3, DMSO treated, LPS stimulated RAW264.7 cells
|
| Data processing |
Background correction by the normexp method; normalisation within arrays by Loess method and between arrays by Scale method; dyeswap correction; statistical analysis by RankProd method
|
| |
|
| Submission date |
Aug 23, 2017 |
| Last update date |
Mar 21, 2018 |
| Contact name |
RACHEL SIMMONDS |
| E-mail(s) |
rachel.simmonds@surrey.ac.uk
|
| Phone |
01483684714
|
| Organization name |
University of Surrey
|
| Street address |
FHMS UNIVERSITY OF SURREY, STAG HILL CAMPUS
|
| City |
GULDFORD |
| State/province |
Surrey |
| ZIP/Postal code |
GU2 7XH |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL10333 |
| Series (1) |
| GSE103002 |
LPS stimulated murine RAW264.7 cells, incubated with DMSO carrier or mycolactone |
|
