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| Status |
Public on Sep 25, 2019 |
| Title |
Splenic_nonsti_iEm_2 |
| Sample type |
SRA |
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| Source name |
Splenic mature B cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Splenic mature B cells
genotype: AID-deficient primary splenic B cells
activation: none
treatment: none
target locus: AID-deficient primary splenic B cells
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| Treatment protocol |
Purified B cells were stimulated with either aCD40/IL4 for 48 hrs; CH12F3 cell lines were cultured in lymphocyte medium R15 and stimulated with anti-CD40+IL4+TGF-β for 24 hrs.
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| Growth protocol |
RPMI1640+10% FBS
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
About 10 million cells were crosslinked with 2% formaldehyde for 10 minutes at room temperature and quenched with glycine at a final concentration of 125 mM. Then, the crosslinked cells were lysed in the 3C lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, protease inhibitors) and nuclei were digested with NlaIII enzyme (NEB, R0125) at 37 ℃ overnight. They were then brought to 16℃, 100U of T4 ligase added (Promega, M1801) and they incubated again overnight at 16℃. The ligated products were de-crosslinked with Proteinase K (Roche, #03115852001) at 56 ℃ overnight and the 3C templates were purified by phenol/chloroform.
Libraries were prepared using 3C samples by HTGTS (Jane et al., 2018).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Data processing |
Library strategy: 3C-HTGTS
Standard basecalling formats for Miseq reads
Miseq reads were de-multiplexed and adapter sequence trimmed using the fastq-multx tool from ea-utils (http://code.google.com/p/eautils/) and the SeqPrep utility (https://github.com/jstjohn/SeqPrep) respectively.
Reads were mapped to the mm9 reference genome using Bowtie2 (http://bowtiebio.sourceforge.net/bowtie2/manual.shtml) with the top fifty alignments reported that had an alignment score above 50, representing a perfect 25nt local alignment.
We used a best-path searching algorithm to select the optimal sequence of alignments that describe the read’s composition. Aligned reads were filtered on the following conditions: (1) reads must include both a bait alignment and a prey alignment and (2) the bait alignment cannot extend more than 10 nucleotides beyond the targeted site. For vector controls and offset nicking with multiple sites, the longest targeted site was used.
We compared discarded alignments to the selected prey alignment; if any of the discarded alignments surpassed both a coverage and score threshold with respect to the prey alignment, the read was filtered due to low mapping quality.
To remove possible mispriming events and other artifacts, the bait alignment must extend 10 nucleotides past the primer.
Post-filter stringency was applied to remove background-prone junctions with gaps larger than 30nt and bait sequences shorter than 50nt.
Genome_build: mm9
Supplementary_files_format_and_content: bedgraph files were generated for displaying (Jane et al., 2018)
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| Submission date |
Apr 24, 2019 |
| Last update date |
Sep 26, 2019 |
| Contact name |
Frederick W Alt |
| E-mail(s) |
jianqiao.hu@childrens.harvard.edu
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| Organization name |
Boston Children's Hospital
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| Department |
PCMM
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| Lab |
Alt
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| Street address |
1 Blackfan Circle
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL16417 |
| Series (2) |
| GSE130263 |
Chromatin Loop Extrusion Plays a Fundamental Mechanistic Role in Antibody Class Switching [3C-HTGTS] |
| GSE130270 |
Chromatin Loop Extrusion Plays a Fundamental Mechanistic Role in Antibody Class Switching |
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| Relations |
| BioSample |
SAMN11491166 |
| SRA |
SRX5730078 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3734790_Splenic_nonsti_iEm_2.bedgraph.gz |
488.8 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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