|
| Status |
Public on Feb 27, 2020 |
| Title |
Bcell_GROSeq_Spt5dep2_24474 |
| Sample type |
SRA |
| |
|
| Source name |
primary activated splenic B lymphocytes
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Supt5hFl/- Rosa26ERT2-cre/+ primary mature B cells
treatment: 4-hydroxy tamoxifen (4-HT)
|
| Treatment protocol |
Primary acttivated B cells were activated for 28h prior to the addition of 2 uM 4-HT for 32h (total of 60 h B cell activation with 32h 4-HT treatment) and then harvested for all downstream applications.
|
| Growth protocol |
Primary immature B cells were isolated from spleens of Supt5hFl/-Rosa26ERT2-cre/+ and Rosa26ERT2-cre/+ mice and cultured in complete RPMI medium wit IL4/LPS stimulation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Following cell membrane lysis with hypotonic buffer, nuclei were isolated via sucrose gradient density centrifugation. GROseq was performed as described (Core & Lis, 2008). Briefly, 10 million nuclei were used per reaction, and the run-on was performed for 5 minutes with NTPs with a limiting concentration of P32-dCTP and dBrdUTP followed by extraction of RNA with Trizol reagent (Thermo Fisher, 15596026). An updated protocol was kindly provided by Dr. John Lis (Cornell University).
After base hydrolysis and DNA end repair, Illumina compatible 3' RNA adapters were ligated to total RNA. After BrdU- incorporated RNA IP the 5' cap was removed and the 5' DNA end was repaired. Next, the 5' DNA Illumina comaptible adapters were ligated, and the library was reverse transcribed and amplified.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Library strategy: GRO-seq
Adapters were trimmed from the 3' ends of the reads with cutadapt (v 1.10), and a 5nt random linker was removed from the 3' end using fastx_trimmer.
Trimmed reads larger than 10nt were aligned to the genome using bowtie v1.0.0 (-v 2 --best --strata -- tryhard -m 1).
Genome_build: NCBI mm9
Supplementary_files_format_and_content: bigWig of rpm-normalized read densities
|
| |
|
| Submission date |
May 31, 2019 |
| Last update date |
Feb 27, 2020 |
| Contact name |
Tobias Neumann |
| Organization name |
IMP
|
| Street address |
Campus-Vienna-Biocenter 1
|
| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE132029 |
Regulation of enhancer transcription by Spt5 directly couples enhancer activation with enhancer function |
|
| Relations |
| BioSample |
SAMN11909956 |
| SRA |
SRX5938652 |
