|
| Status |
Public on Dec 31, 2013 |
| Title |
SHSY5Y_F |
| Sample type |
RNA |
| |
|
| Source name |
pooled Human SH-SY5Y cells total RNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SH-SY5Y
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples of total RNA from Human tissues were purchased from several companies (Clontech, Ambion, Stratagene, Cell Applications). HeLa and SH-SY5Y cells were cultured in standard DMEM / 10% FBS medium. Total RNA was extracted from cells with TRIzol regent (Invitrogen). Additionally, total RNA was purified with Rneasy Mini Kit (Qiagen) and treated with RNase-free DNase Set (Qiagen) to remove any contaminating genomic DNA.
|
| Label |
biotin
|
| Label protocol |
Reverse transcription, amplification, fragmentation and labeling steps were performed according to Affymetrix protocol using WT Amplified Double-Strand cDNA Synthesis Kit (Affymetrix) and WT Double-Strand DNA Terminal Labeling Kit (Affymetrix).
|
| |
|
| Hybridization protocol |
Approximately 6.0μg of DNA was hybridized per array using the Affymetrix hybridization kit. For these 14 array sets, 7 arrays were hybridized for 16 hours at 45°C at 60rpm using an Affymetrix hybridization oven. The hybridized solution was removed from these arrays and reused to hybridize the remaining seven arrays.
|
| Scan protocol |
Arrays were scanned on an Affymetrix Scanner 3000 7G. Images were quantified using GCOS software (Affymetrix). Signal values ware exported to text files using GCOS Manager software (Affymetrix).
|
| Description |
SH-SY5Y cells Human Tiling 1.0R chip F
|
| Data processing |
The ".CEL" files are raw data. The "normalized.txt" files (supplementary data linked to GSE18490) are data normalized as follows.
1) To exclude potential low quality probes, we excluded probes that corresponded to the following conditions: A) probes for which either the Perfect Match (PM) or Miss Match (MM) probe perfectly match more than once in the genome, B) probes which contain SNPs, C) probes located on sex chromosomes.
2) To reduce inter-chip differences, we equalized the mean and variance of probe intensities among all chips in each tissue. Note that the intensities were converted to a logarithmic scale (base 2).
3) To reduce background noise, we subtracted the intensity of each MM probe from the PM probe in each probe set. If the value was negative, zero was assigned as the value.
4) To adjust for GC content of probes, we calculated normalized intensities by each GC content.
5) To equalize the distribution of signals in each chip, we performed quantile normalization for each chip
6) To smooth the probe intensities with neighboring probes, we calculated Hodges-Lehmann statistics. The size of each sliding window was 101bp.
7) All probes that ranked higher than the top 15% signal threshold were selected and those with an inter-probe distance less than a maximum gap threshold of 25bp were concatenated. Then, the concatenated probes with length longer than a minimum run threshold of 101bp were defined as transfrags. Finally, we performed BLAST to exclude probes that mapped to multiple locations.
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| |
|
| Submission date |
Oct 09, 2009 |
| Last update date |
Dec 31, 2013 |
| Contact name |
Fuyuki Miya |
| E-mail(s) |
fmiya@keio.jp
|
| Phone |
+81-3-5363-3890
|
| Organization name |
Keio University
|
| Department |
School of Medicine
|
| Lab |
Center for Medical Genetics
|
| Street address |
35 Shinanomachi, Shinjuku-ku
|
| City |
Tokyo |
| State/province |
Tokyo |
| ZIP/Postal code |
160-8582 |
| Country |
Japan |
| |
|
| Platform ID |
GPL6161 |
| Series (2) |
| GSE18490 |
Whole genome transcriptional profile of human tissues and cells |
| GSE18676 |
Whole genome expression in human tissues |
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