|
| Status |
Public on Sep 27, 2013 |
| Title |
Resting B cells Replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
CD43 negative mouse resting B cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
genotype/variation: wild type
developmental stage: adult
tissue: Spleen
cell type: CD43 negative mouse resting B cells
|
| Treatment protocol |
Resting B cells were isolated from the spleens of adult mice. The spleens were dissected and minced on sieves and the single-cell suspensions were centrifuged on a Ficoll-Paque cushion to remove erythrocytes and dead cells. The resulting splenocytes were resuspended at a concentration of 10^7 cells/ml in 0.1% BSA/HBSS supplemented with 5% goat serum and anti-CD16 antibody. The cells were firstly incubated with biotinylated anti-CD43 antibody and then with Dynabeads Biotin Binder beads to capture CD43+ cells. Bound CD43+ cells were removed by 4 sequential magnetic separations. The remaining population of CD43- cells was cultured at a concentration of 2-5x10^6 cells/ml.
|
| Growth protocol |
RPMI, 15% foetal calf serum, 0.1 U/mL penicillin, 0.1 μg/mL streptomycin, 2 mM L-Glutamine, 50 μM beta-mercaptoethanol, 6 ng/ml IL-4
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell lysates were homogenized using QIAshredder spin columns (Qiagen). Total RNA was purified with RNeasy Mini Kit (Qiagen), eluted in 30 μl of DEPC-treated water and quantified on an ND-1000 spectrophotometer. mRNA-seq libraries were prepared using 2 μg of total RNA purified as previously described. A total of four libraries were prepared for four independent biological replicates. Libraries were prepared using the TruSeq RNA Sample Preparation Kits v2 (Illumina) according to the manufacturer’s protocol.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Expression profiling of Quiescent B-cells
|
| Data processing |
RNA-seq sequencing reads were aligned to mm9 using Tophat version 1.4.1 with default parameters. Gene coordinates from Ensembl version 64 was used as known gene model
FPKM values for each sample was estimated using Cufflinks version 1.3.0
Genome_build: mm9
Supplementary_files_format_and_content: FPKM values
|
| |
|
| Submission date |
Dec 04, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Gopuraja Dharmalingam |
| Organization name |
MRC London Institute of Medical Sciences
|
| Department |
Epigenetics section
|
| Street address |
Faculty of Medicine, Imperial College, Hammersmith Hospital Campus
|
| City |
London |
| ZIP/Postal code |
W12 0NN |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE42704 |
The Aurora B kinase and the polycomb protein Ring1B combine to regulate active promoters in quiescent lymphocytes [RNA-Seq] |
| GSE42706 |
The Aurora B kinase and the polycomb protein Ring1B combine to regulate active promoters in quiescent lymphocytes |
|
| Relations |
| SRA |
SRX208220 |
| BioSample |
SAMN01822818 |
