|
| Status |
Public on Jan 08, 2018 |
| Title |
Lig4-/- clone #2_c-myc_5'BE_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
CH12F3 murine lymphoma cells
|
| Organism |
Mus musculus |
| Characteristics |
genotype: Lig4-/- CH12F3 clone #2
activation: aCD40/IL4/TGFb
treatment protocol: cytokine stimulation + c-myc gRNA/Cas9
target locus: c-myc gRNA/Cas9 induced bait DSB
growth conditions: RPMI1640; 15% FBS
|
| Treatment protocol |
Purified B cells were stimulated with either aCD40/IL4 for 96 hrs; CH12F3 cell lines were cultured in lymphocyte medium R15 and stimulated with anti-CD40+IL4+TGF-β for 72 hrs.
|
| Growth protocol |
RPMI1640+15% FBS
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted overnight at 55 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 HTGTS.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
| |
|
| Data processing |
Library strategy: 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.
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 |
Nov 15, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Rohit A Panchakshari |
| E-mail(s) |
rohit.panchakshari@childrens.harvard.edu
|
| Organization name |
Children's Hospital Boston
|
| Department |
PCMM
|
| Lab |
Frederick W. Alt Lab
|
| Street address |
One Blackfan Circle, KARP building
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL16417 |
| Series (1) |
| GSE106922 |
DNA Double-strand Break Response Factors Influence End-joining Features of IgH Class Switch and General Translocation junctions |
|
| Relations |
| BioSample |
SAMN08028310 |
| SRA |
SRX3394609 |
