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Status |
Public on Jan 31, 2017 |
Title |
SHM_Ubc_Duvelisib rep1 |
Sample type |
SRA |
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Source name |
CSR activated B cells
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Organism |
Mus musculus |
Characteristics |
strain: 129 tissue: spleen age: 8-12 weeks genotype: WT cell type: splenic B cells activated with anti-CD40 plus IL-4 for 4 days treatment: duvelisib
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Treatment protocol |
1 micromolar Duvelisib
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Growth protocol |
Splenic naïve B cells were purified via an anti-CD43 negative selection approach. The purified naïve B cells were cultured in RPMI 1640 medium supplemented with 10% FBS and activated with anti-CD40 plus IL-4 for 4 days to generate CSR-activated B cells, and treated with duvelisib.
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Extracted molecule |
genomic DNA |
Extraction protocol |
Cells were lysed in lysis buffer (NaCl 200 mM, Tris.HCl 100 mM, EDTA 5mM, SDS 0.2%) containing 10 μg/ml Proteinase K at 56°C overnight. Genomic DNA was precipitated with isopropanol at room temperature, washed in ethanol 70% and resuspended in TE buffer (10mM Tris-HCl plus 1mM EDTA) Phusion High Fidelity DNA polymerase was used to amplify selected regions from template genomic DNA. Amplification products were purified using PCR purification kit and GEL extraction kit and sequenced bi-directionally in a Mi-Seq Illumina sequencing platform
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina MiSeq |
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Data processing |
For SHM data, sequences with mean quality score < 20 and length < 50 were removed. The remained sequences were used to calculate mutation rate. Sequences obtained from each designed region were aligned to the reference sequence using BLASTN with alignment length > 200. Mutations to be considered valid were calculated after filtering steps, as previously described(Liu, M. et al.,Nature 2008). Briefly, mutations first had to pass a Neighbourhood Quality Standard (NQS) criteria requiring a minimum Phred score of 30 for the mutation itself, and 20 for the five adjacent bases on either side. Mutations that were within five bases of more than two additional mutations were excluded. Mutations within two bases of a deletion or insertion were also excluded. In addition, bases with mutation rate > 0.01 were excluded as a result of overwhelming influence of sequence error or SNP, of which bases with mutation rate > 0.2 were further regarded as SNP and were excluded. Finally, the average base mutation rate of 1-200 bp passing the above criteria were calculated from forward sequence, as well as reverse sequences. For average base mutation rates of C-to-T or G-to-A transitions, only C or G bp sites we counted.
Supplementary_files_format_and_content: For SHM data, tab-delimited text files give mutation information for each base pair incuding mut frequency for all mut, particular C>T|G>A and others. Bases to fail to pass filter criteria were showen as 'NA'.
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Submission date |
Feb 10, 2016 |
Last update date |
May 15, 2019 |
Contact name |
Roberto Chiarle |
E-mail(s) |
Roberto.Chiarle@childrens.harvard.edu
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Organization name |
Children’s Hospital Boston and Harvard Medical School
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Department |
Department of Pathology
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Lab |
Roberto Chiarle
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Street address |
300 Longwood Avenue
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City |
Boston |
State/province |
Massachusetts |
ZIP/Postal code |
02115 |
Country |
USA |
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Platform ID |
GPL16417 |
Series (1) |
GSE77788 |
Phosphatidylinositol 3-Kinase (PI3K) delta blockade increases genomic instability in B cells |
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Relations |
BioSample |
SAMN04485232 |
SRA |
SRX1570187 |
Supplementary file |
Size |
Download |
File type/resource |
GSM2059377_SHM_Ubc_Duvelisib_1.BMF.txt.gz |
1.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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