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| Status |
Public on Sep 13, 2017 |
| Title |
LPS-Etoposide 2 48-4 h pf 7_Replicate 2 |
| Sample type |
SRA |
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| Source name |
LPS-Etoposide 2 48-4 h pf 7
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6Babr
cell type: primary, splenic B cells
stimulation: LPS (48 hours) + Etoposide (4 hours)
cell fraction: Polysomes
fraction id: Polysome fraction 7
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| Treatment protocol |
After isolation, splenic B cells were cultured in RPMI 1640 Medium (Dutch Modification) plus 5% FCS, antibiotics, 2 mM L-glutamine and Beta-mercaptoethanol (5 microMolar) at a density of 5x10^5 cells/ml. They were activated with LPS (10 micrograms/ml) for 47 hours prior adding the ATM kinase inhibitor KU55933 (10 microMolar) for 1 hour. Then, DNA damage was induced with etoposide (20 microMolar) for 4 hours. Cells were then treated with cycloheximide (100 microgrames per ml) for 3 minutes prior isolation of cytoplasmic extracts and polysome fractionation using sucrose gradients (10-30%). 1 ml fractions containing monosomes (fractions 4 to 7), light polysomes (fractions 8 to 10) or heavy polysomes (fractions 11 to 16) were collected .
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| Growth protocol |
Splenic B cells from C57BL6_Babr mice were isolated using a B cell isolation kit from Miltenyi Biotech. Mice were maintained under Babraham Institute AWEEC and UK home Office regulation.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
mRNA was precipitated using 8M guanidine hydrochloride and absolute ethanol
Custom protocol (described in the associated publication).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1000 |
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| Data processing |
Sample demultiplexing (39-40 samples per lane) based on Illumina first read (10 bp; not provided).
Location and length analysis of the longest consecutive A tract in raw reads (20 nucleotide long A tract expected). Removal of the A-tract from reads was performed according to the following rules (tail length, A tract length, allowed mismatches in tract): (20, 17, 2), (19, 16, 2), (18, 15, 2), (17, 14, 2), (16, 13, 1), (15, 12, 1), (14, 11, 1), (13, 10, 1), (12, 10, 1), (11, 9, 1), (10, 8, 1), (9, 8, 1), (8, 7, 0), (7, 6, 0), (6, 5, 0), (5, 3, 0), (4, 3, 0).
Reads containing internal A-tracts are cut according to rule (minimal A tract length, allowed mismatches in tract): (17, 2). Remove any remaining A from the end of read.
Reads were mapped to GRCm38/mm10 genome (Ensembl 66.8 annotation)with TopHat (v2.0.12) using tophat -p 6 -g 2 parameters.
Read counting was performed using HT-Seq (Mus_musculus.GRCm38.78.gtf annotation)
Differential gene expression analysis was performed using DESeq2_1.6.3 package. (GLM; read.counts ~ condition + polysome.fraction + condition:polysome.fraction)
Genome_build: mm10
Supplementary_files_format_and_content: Read counts
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| Submission date |
Jan 12, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Felix Krueger |
| E-mail(s) |
fkrueger@altoslabs.com
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| Organization name |
Altos Labs
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| Department |
Bioinformatics
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| Street address |
Granta Park
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| City |
Cambridge |
| ZIP/Postal code |
CB21 6GP |
| Country |
United Kingdom |
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| Platform ID |
GPL15103 |
| Series (2) |
| GSE93573 |
Regulation of mRNA translation and subcellular location controls protein synthesis of key modulators of the DNA damage response during B cell activation [PolyRiboSeq] |
| GSE93576 |
Regulation of mRNA translation and subcellular location controls protein synthesis of key modulators of the DNA damage response during B cell activation |
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| Relations |
| BioSample |
SAMN06226801 |
| SRA |
SRX2487742 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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