|
| Status |
Public on Nov 21, 2013 |
| Title |
n2a-pax6_control-R2 |
| Sample type |
RNA |
| |
|
| Source name |
N2A_non-targeting vector
|
| Organism |
Mus musculus |
| Characteristics |
cell line: neuro 2A (N2A)
cell type: neuroblastoma
transiently transfected with: non-targeting control vector
|
| Treatment protocol |
Approximately 2x10^5 cells were plated per well in a 6 well plate. 16-24 hrs later cells were transfected with 1.5 ug shRNA expression construct using FuGENE 6 (Promega) according to the manufacturer’s instructions. Total RNA was extracted from the cells 3 days later.
|
| Growth protocol |
N2A mouse neuroblastoma cells were grown in DMEM supplemented with 10% fetal bovine serum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the Qiagen Mini RNeasy kit following the manufacturer’s instructions and RNA integrity assessed on a BioAnalyzer (Agilent Technologies).
|
| Label |
biotin
|
| Label protocol |
200 ng RNA was used to generate labelled sense single stranded DNA (ssDNA) for hybridization with the Ambion WT Expression Kit, the Affymetrix WT Terminal Labelling and Controls Kit and the Affymetrix Hybridization, Wash, and Stain Kit as described by the manufacturer. Sense ssDNA was fragmented and the distribution of fragment lengths was measured on a BioAnalyzer.
|
| |
|
| Hybridization protocol |
Fragmented ssDNA was then labelled and hybridized to the Affymetrix GeneChip Mouse Gene 1.0 ST Array (Affymetrix). Chips were processed on an Affymetrix GeneChip Fluidics Station 450.
|
| Scan protocol |
Chips were scanned on an Affymetrix Scanner 3000.
|
| Description |
control2
|
| Data processing |
Affymetrix CEL files were analysed using the Limma, oligo, and genefilter R Bioconductor packages. Arrays were RMA background corrected, quantile normalised and summary expression values calculated for Refseq and full length mRNAs. Before differential expression analysis, genes changing upon Pax6 knockdown were filtered to remove genes genes with consistently low expression.
|
| |
|
| Submission date |
Nov 20, 2013 |
| Last update date |
Nov 21, 2013 |
| Contact name |
Stephen Sansom |
| E-mail(s) |
stephen.sansom@kennedy.ox.ac.uk
|
| Organization name |
Kennedy Institute of Rheumatology
|
| Department |
NDORMS
|
| Lab |
Sansom
|
| Street address |
Roosevelt Drive
|
| City |
Oxford |
| State/province |
Oxfordshire |
| ZIP/Postal code |
OX3 7FY |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL6246 |
| Series (2) |
| GSE52568 |
The long non-coding RNA Paupar regulates the expression of both local and distal genes [Pax6 KD] |
| GSE52571 |
The long non-coding RNA Paupar regulates the expression of both local and distal genes |
|
