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Status |
Public on Jun 28, 2017 |
Title |
nsRNA_seq_T_NT_2 |
Sample type |
SRA |
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Source name |
primary T cells
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Organism |
Mus musculus |
Characteristics |
cell type: primary T cells genotype: WT strain: C57BL/6
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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
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
NextSeq 550 |
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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
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Submission date |
May 23, 2017 |
Last update date |
May 15, 2019 |
Contact name |
Yaakov Maman |
Organization name |
Bar-Ilan University
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Department |
Azrieli Faculty of Medicine
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Lab |
Genome Instability & Cancer
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Street address |
Henrietta Szold 8
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City |
Safed |
ZIP/Postal code |
1311502 |
Country |
Israel |
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Platform ID |
GPL21626 |
Series (2) |
GSE99196 |
Genome Organization Drives Chromosome Fragility [nsRNA-seq] |
GSE99197 |
Genome Organization Drives Chromosome Fragility |
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Relations |
BioSample |
SAMN07156402 |
SRA |
SRX2843878 |
Supplementary file |
Size |
Download |
File type/resource |
GSM2635616_nsRNA_seq_T_NT_2.bw |
143.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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