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| Status |
Public on Dec 14, 2022 |
| Title |
DRIP-seq rnaseh2b-/- setx-/- biol rep 1 |
| Sample type |
SRA |
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| Source name |
Spleen
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| Organism |
Mus musculus |
| Characteristics |
tissue: Spleen
cell type: primary B cells
genotype: rnaseh2b-/- setx-/-
treatment: LPS/a-RP105/IL-4
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| Treatment protocol |
CD43- resting B cells were isolated and cultured in B cell media (BCM, RPMI-1640 supplemented with 10% fetal calf serum, 1% L-glutamine, 50 IU/ml penicillin/streptomycin, 1% sodium pyruvate, 53 μM 2-mercaptoethanol, 10 mM HEPES).
B cells were stimulated with LPS, α-RP105 and interleukin 4 (IL-4) for IgG1, LPS/α-RP105/TGF-B for IgG2b and LPS/α-RP105/TGF-B/CD40L for IgA class switch recombination.
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| Growth protocol |
Setx-/- , Rnaseh2bf/f, and CD19cre mice were previously described and used to generate Setx-/- Rnaseh2bf/f CD19cre mice (Becherel et al., 2013; Hiller et al., 2012; Rickert et al., 1997)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For LAMS-HTGTS, genomic DNA was isolated from WT, Setx-/- , Rnaseh2bf/f , and Setx-/- Rnaseh2bf/f cells 72 hours after stimulation to switch to IgG1 with 72 h LPS/IL-4/α-RP105.
For DRIP-seq, genomic DNA was gently extracted by SDS/Proteinase K treatment at 37°C followed by phenol-chloroform extraction and ethanol precipitation. The extracted genomic DNA was fragmented with restriction enzyme (Hindlll, Xbal, EcoRI, SspI, BrsGI), 4 micrograms of digested DNA were incubated with 2 micrograms of S9.6 antibody overnight at 4 ℃ in DRIP buffer (10 mM NaPO4 pH 7.0, 140 mM NaCl, 0.05% Triton X-100). In vitro RNase H digestion was used to generate a negative control. After incubation, antibody-DNA complexes were bound to Protein G Dynabeads and thoroughly washed, DNA was recovered with Chelex-100, the bound fraction was suspended in 0.1 ml 10% Chelex-100 (Bio-Rad), vortexed, boiled for 10 min, and cooled to room temperature. This sample was added to 4 μl of 20 mg/ml Proteinase K followed by incubation at 55°C for 30 min while shaking. Beads were boiled for another 10 min. Sample was centrifuged and supernatant collected. Beads were suspended with 100 μl 2× TE to the beads, vortexed, centrifuged, and supernatants pooled.
For LAMS-HTGTS, linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) libraries were prepared from genomic DNA as previously described (Hu et al., 2016; Yin, Liu, Liu, & Hu, 2019; Yin, Liu, Liu, Wu, et al., 2019).
For DRIP-seq, libraries were prepared as described in (Ginno et al., 2012; Sanz et al., 2016)
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
For LAMS-HTGTS, sequences were processed and analyzed as detailed in (Crowe et al., 2018; Hu et al., 2016; https://github.com/robinmeyers/transloc_pipeline) with minor adjustments. Briefly, sequences were aligned to custom genomes, substituting the mm9 sequence from (114, 494, 415–114, 666, 816) with sequence from the NG_005838.1 (Genbank accession no. NG_005838.1) C57BL/6 IgH sequence on chr 12 (to 11,172 to 183,818), or sequence from the AJ851868.3 (GenBank accession no AJ851868.3) 129 IgH sequence (1,415,966– 1,592,715).Junctions were analyzed by comparing sequences to both C57BL/6 and S129 backgrounds. A consensus genome was generated between C57BL/6 and S129, and if reads fell into either category they were deemed not mutated—this eliminated over-estimation of insertions, deletions, and mismatch mutations. Of note, no junctions aligned to S129 on one side and C57BL/6. Deletions are defined as regions missing nucleotides adjacent to prey-break site but having 100% homology in flanking regions. Insertions are defined as regions containing nucleotides that map to neither the bait nor the prey-break site. Microhomologies (MHs) are defined as regions of 100% homology between the bait and the prey-break site. Blunt junctions are considered to have no MHs or insertions. Additional scripts used to analysis can be found in github (https://github.com/srhartono/TCseqplus).
For DRIP-seq, raw sequencing reads were trimmed with fastq-mcf v2.4.4 and mapped to mm9 genome with bowtie2 v2.2.6. Wig files were generated using bedtools genomecov and converted into bigWig using wigToBigWig; scores indicate tag per million (x30). Peak bed files were generated using HMM as previously described in (Sanz et al., 2016) and converted into bigBed using bedToBigBed.
Assembly: mm9
Supplementary files format and content: bigWig, bigBed, and tlx (tab-separated files containing translocation coodinates and information)
Library strategy: DRIP-Seq
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| Submission date |
Apr 21, 2022 |
| Last update date |
Dec 17, 2022 |
| Contact name |
Frederic Chedin |
| E-mail(s) |
flchedin@ucdavis.edu
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| Organization name |
UC Davis
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| Department |
MCB
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| Lab |
Chedin
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| Street address |
1 Shields Avenue
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| City |
Davis |
| State/province |
CA |
| ZIP/Postal code |
95616 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (1) |
| GSE201210 |
Senataxin and RNase H2 act redundantly to suppress genome instability during class switch recombination |
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| Relations |
| BioSample |
SAMN27720013 |
| SRA |
SRX14951505 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6052747_LS99C_S24_L008_R1_001.bigBed |
6.7 Mb |
(ftp)(http) |
BIGBED |
| GSM6052747_LS99C_S24_L008_R1_001.bigWig |
142.2 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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