|
| Status |
Public on Jul 03, 2008 |
| Title |
Transcriptome of B cells |
| Sample type |
RNA |
| |
|
| Source name |
ramos B cells
|
| Organism |
Homo sapiens |
| Characteristics |
Ramos B cell line
|
| Growth protocol |
B cells were cultured parallely in 2*150 cm3 flasks with RPMI 1640 (penicillin-streptomycin) (Gibco) supplemented with 10% FCS (Biochrom) and Glutamax (1x) (Gibco). At passage 20 (confluent state), cells were trypsinized, washed off in PBS, pelleted, and resuspended in 2 ml lysis solution RLT (Qiagen).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA preparation and double stranded cDNA generation
Total RNA was then extracted from ~ 20 x 106 cells per sample (2*HEK and 2*B cells samples) using RNeasy Midi extraction kit (Qiagen) by following the manufacturer’s instructions. DNA was removed using the “on column digestion” protocol of the RNeasy Midi extraction kit (Qiagen). Total RNA quality was assessed by spectrophotometry (Nanodrop) and gel electrophoresis (1% agarose).
|
| Label |
Biotin
|
| Label protocol |
Biotin-labelled cRNA was generated using a linear amplification kit (Ambion #IL1791) starting with 500 ng of DNA-free, quality-checked total RNA of each sample as input (see RNA preparation).
|
| |
|
| Hybridization protocol |
Chip hybridisations, washing and Cy3-streptavidin (Amersham Biosciences) staining were performed on the Illumina BeadStation 500 platform employing reagents and following protocols supplied by the manufacturer.
|
| Scan protocol |
scanning was performed on the Illumina BeadStation 500 platform
|
| Description |
cRNA samples were hybridized as biological and technical duplicates on Illumina HumanRef8 V2.0 BeadChips.
B1-1raw.txt: B cell biological replicate 1, technical replicate 1
B1-2raw.txt: B cell biological replicate 1, technical replicate 2
B2-1raw.txt: B cell biological replicate 2, technical replicate 1
B2-2raw.txt: B cell biological replicate 2, technical replicate 2
|
| Data processing |
Raw expression data (BeadSummary gene profile files), without normalization, were extracted from the manufacturer’s Illumina BeadStudio application software 1.0. Further processing of the data was done within the R / Bioconductor statistical analysis software package. We calculated the Pearsons product moment correlation coefficient on the non-normalized data sets to remove outliers and to check for integrity of the experiments (r>=0.98), followed by the computation of intra-chip pair-wise scatter-plots for each probe. The raw data sets were then normalized using quantile normalization.
|
| |
|
| Submission date |
Jun 26, 2008 |
| Last update date |
Aug 18, 2008 |
| Contact name |
Marc Sultan |
| E-mail(s) |
[email protected]
|
| Organization name |
Max Planck Institute for molecular genetics
|
| Street address |
Ihnestr. 73
|
| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
| |
|
| Platform ID |
GPL6104 |
| Series (1) |
| GSE11892 |
A Global View of Gene Activity and Alternative Splicing by Deep Sequencing of the Human Transcriptome |
|
