|
| Status |
Public on Jun 28, 2017 |
| Title |
nsRNA_seq_TRPexp_aB_TRIPTOLIDE |
| Sample type |
SRA |
| |
|
| Source name |
Activated primary B cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Activated primary B cells
genotype: WT
strain: C57BL/6
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
B cell cultures stimulated for 12 hours and T cell cultures for 4 hours. Resting B cells were incubated in media without stimulation. 4 million labeled with 0.5 mM 5-ethynyl uridine (EU) for 30 minutes at 37°C. For B-cells DRB (D-ribofuranosylbenzimidazole) (150 μM) or Triptolide (3 μM) was added to 90 minutes prior to EU labeling, and etoposide was present in the medium 5 minutes before and during the EU labeling (30 minutes). Total RNA extraction was performed using TRIzol (Ambion) and 1 ug of RNA as used. Ribosomal RNA was depleted using the NEBNext rRNA Depletion kit (human/mouse/rat) (New England Biosciences), prior to biotinylation by the Click-it reaction (Click-iT Nascent RNA Capture Kit, ThermoFisher C10365). First-strand cDNA synthesis of the captured nascent RNA was done using SuperScript VILO cDNA synthesis kit (Invitrogen), second-strand cDNA was synthesized with 10 mM dNTPs in the presence of RNase H, E. coli DNA polymerase I (Invitrogen) and sequencing libraries were prepared following the TruSeq RNA sample prep protocol (Illumina).
libraries were prepared and sequenced following standard Illumina protocols
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
| |
|
| Data processing |
Library strategy: nascent RNA-seq
Reads were aligned to the GRCm38p2/mm10 genome assembly
Alignment done using bowtie version 0.12.7, with the parameters: --best --all --strata -n 3 -m1 -l 50
Peaks were called using the parameters: --nolambda,-- nomodel.
Genome_build: GRCm38p2/mm10
Supplementary_files_format_and_content: bigwig files were made using Bedtools genomecov function followed by UCSC toolkit function bedGraphToBigWig
|
| |
|
| Submission date |
May 23, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Yaakov Maman |
| Organization name |
Bar-Ilan University
|
| Department |
Azrieli Faculty of Medicine
|
| Lab |
Genome Instability & Cancer
|
| Street address |
Henrietta Szold 8
|
| City |
Safed |
| ZIP/Postal code |
1311502 |
| Country |
Israel |
| |
|
| Platform ID |
GPL21626 |
| Series (2) |
| GSE99196 |
Genome Organization Drives Chromosome Fragility [nsRNA-seq] |
| GSE99197 |
Genome Organization Drives Chromosome Fragility |
|
| Relations |
| BioSample |
SAMN07156398 |
| SRA |
SRX2843882 |
