|
| Status |
Public on Sep 25, 2019 |
| Title |
IaD-i3CBEc_2 |
| Sample type |
SRA |
| |
|
| Source name |
CH12F3 murine lymphoma cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: CH12F3 murine lymphoma cells
genotype: Ialpha-delta-i3CBEc
activation: aCD40/IL4/TGFb
treatment: cytokine stimulation
target locus: three CBEs insertion downstream of Cg2a in Ia deleted cells
|
| Treatment protocol |
CH12F3 cell lines were cultured in lymphocyte medium R15 and stimulated with anti-CD40+IL4+TGF-β for 72 hrs.
|
| Growth protocol |
RPMI1640+10% FBS
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted overnight at 56 degree Celcius in a lysis buffer containing: 200mM NaCl, 0.4% SDS, 100mM Tris-HCl pH7.5, 5mM EDTA pH8.0, 200ug/mL proteinase K, prior to DNA fragmentation
Translocation libraries were prepared using CSR-HTGTS-Seq (Dong et al., 2015)
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
Library strategy: CSR-HTGTS-Seq
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.
We removed potential duplicates by comparing the coordinates of the end of the bait alignment and the start of the prey alignment across all reads. A read will be marked as a duplicate if it has a bait alignment offset within 2nt and a prey alignment offset within 2nt of another read’s bait and prey alignments.
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: tab delimited text files contain filtered unique junctions and include the following information: sequence ID (Qname); prey chromosome (Rname), prey junction coordinate (Junction), chromosome orientation of prey junction (Strand), beginning (Rstart) and end (Rend) nucleotide position of prey junction aligning to the genome build; bait chromosome (B_Rname); beginning (B_Rstart) and end (B_Rend) nucleotide position of the bait junction; chromosome orientation of the bait junction (B_Strand); position on the read where the bait sequence begins (B_Qstart) and ends (B_Qend); position on the read where the prey junction begins (Qstart) and ends (Qend); the length of the combined and stitched paried end read (Qlen); the entire stitched paired end read sequence (Seq); the sequence in the + orientation at the junction (J_seq); and the experiment associated with the read (Library)
|
| |
|
| Submission date |
Apr 24, 2019 |
| Last update date |
Sep 26, 2019 |
| Contact name |
Frederick W Alt |
| E-mail(s) |
jianqiao.hu@childrens.harvard.edu
|
| Organization name |
Boston Children's Hospital
|
| Department |
PCMM
|
| Lab |
Alt
|
| Street address |
1 Blackfan Circle
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL16417 |
| Series (2) |
| GSE130265 |
Chromatin Loop Extrusion Plays a Fundamental Mechanistic Role in Antibody Class Switching [CSR-HTGTS-Seq] |
| GSE130270 |
Chromatin Loop Extrusion Plays a Fundamental Mechanistic Role in Antibody Class Switching |
|
| Relations |
| BioSample |
SAMN11491289 |
| SRA |
SRX5730182 |
