|
| Status |
Public on Dec 11, 2012 |
| Title |
A1KD_2 |
| Sample type |
RNA |
| |
|
| Source name |
HOXA1_knockdown_rep2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: lung fibroblasts
cell line: IMR-90
transfection: HOXA1 knockdown
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from human cell lines or human fat was extracted with Trizol (Invitrogen) and purified via the RNeasy mini kit (Qiagen) according to the manufacturers’ instructions. RNA quality was assessed using an Aglient Bioanalyzer
|
| Label |
cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.3 ug total RNA using the One-Color Low-Input QuickAmp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600 ng of cRNA (specific activity >10.0 pmol Cy3/ug cRNA) per channel per array spot (8 per slide) was fragmented at 60°C for 30 minutes following the manufacturers instructions. On completion of the fragmentation reaction, 1 volume of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Sureprint G3 8x60k gene expression arrays (G4858A-028004) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent)(both without Triton added).
|
| Scan protocol |
Slides were scanned immediately after washing and placement within an Agilent Ozone Barrier Slide cover (Agilent) the Agilent DNA Microarray Scanner (G2505C) using two color scan setting for 8x60k array Scan resolution 3 microns. Scanning was done in both channels and PMT is set to 100% for both Cy3\cy5 channels.
|
| Description |
Gene Expression
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE2-105_Dec08 with the cognate G4858A-028004 grid file to obtain background subtracted and spatially detrended Processed Signal intensities. Processed Signal Intensities were expressed as a ratio to the 75th percentile value (control probes excluded) and median centered for each array and log2 transformed. Only probes tagged by Feature Extraction as above background in all constituents of a sample triplicate or duplicate set in at least one condition were considered for further analysis. After background filtering, probes values were collapsed by the GeneName column from the Feature Extraction output to condense multiple reads of the same gene. To correct for slide batch effects (each slide = 8 arrays) the R ComBat package (Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics 2007) was employed using the universal reference (technical replicates) array on each slide upon which a universal replicate was hybridized along with each condition treating each array as a different batch.
|
| |
|
| Submission date |
May 01, 2012 |
| Last update date |
Dec 13, 2012 |
| Contact name |
Cole Trapnell |
| E-mail(s) |
cole@broadinstitute.org
|
| Organization name |
Harvard University
|
| Department |
Stem Cell and Regenerative Biology
|
| Lab |
John Rinn
|
| Street address |
7 Divinity Ave
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02138 |
| Country |
USA |
| |
|
| Platform ID |
GPL13607 |
| Series (2) |
| GSE37690 |
Differential analysis of HOXA1 in adult cells at isoform resolution by RNA-Seq [Agilent] |
| GSE37704 |
Differential analysis of gene regulation at transcript resolution by RNA-Seq |
|
