|
| Status |
Public on May 18, 2010 |
| Title |
Human Brain Total RNA, 6 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
Human brain total RNA
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: brain
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total or PolyA+ RNA was obtained from Clontech. After DNaseI (GE Healthcare) treatment to remove residual genomic DNA, 10 ug of RNA was incubated with random hexamers at 70C for 10 min, incubated at 15C for 30 min and reverse transcribed to single-stranded cDNA for 2 hours at 42C with Superscript II (Invitrogen). The cDNA was then directly used for second strand synthesis, after which the double-stranded cDNA (ds-cDNA) was purified using PCR purification columns (Qiagen) in combination with the nucleotide cleanup kit protocols.
|
| Label |
biotin
|
| Label protocol |
cDNA was fragmented by nuclease digestion in a solution containing 1X One-Phor-All buffer (GE Healthcare) and DNase I (GE Healthcare) at 37ｰC until the average fragment size was approximately 70 bp. The fragmentation reaction was stopped by incubating at 95C for 10 min. Fragmented samples were biotinylated with GeneChip Labeling reagent (Affymetrix) using terminal deoxynucleotidyl transferase at 37C for 2 hours.
|
| |
|
| Channel 2 |
| Source name |
Human genomic DNA
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: reference
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was obtained from Clontech.
|
| Label |
biotin
|
| Label protocol |
Genomic DNA was fragmented by nuclease digestion in a solution containing 1X One-Phor-All buffer (GE Healthcare) and DNase I (GE Healthcare) at 37C until the average fragment size was approximately 70 bp. The fragmentation reaction was stopped by incubating at 95C for 10 min. Fragmented samples were biotinylated with GeneChip Labeling reagent (Affymetrix) using terminal deoxynucleotidyl transferase at 37C for 2 hours.
|
| |
|
| |
| Hybridization protocol |
Arrays were hybridized in a GeneChip Fluidics Station 450 using standard protocol “EukGE-WS2v5_450”.
|
| Scan protocol |
Arrays were scanned in an Affymetrix 7G scanner.
|
| Description |
HBT-F1
|
| Data processing |
Raw data files were normalized with Affymetrix Tiling Analysis Software (TAS) v1.1. All samples were quantile normalized with a bandwidth of 70, using a genomic DNA hybridization as reference.
|
| |
|
| Submission date |
Dec 02, 2009 |
| Last update date |
Apr 23, 2010 |
| Contact name |
Harm van Bakel |
| E-mail(s) |
harm.vanbakel@mssm.edu
|
| Organization name |
Mount Sinai School of Medicine
|
| Department |
Genetics and Genomic Sciences
|
| Lab |
Bakel Lab
|
| Street address |
One Gustave L. Levy Place, Box 1498
|
| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10029 |
| Country |
USA |
| |
|
| Platform ID |
GPL4915 |
| Series (1) |
| GSE19289 |
Most “dark matter” transcripts are associated with known genes |
|
