What are the best types of samples for BrITL?
Cells must be proliferating – BrITL has been successfully performed on cells that experience S-phase-specific DNA damage
Evidence of a sufficient presence of DNA double-strand breaks in cells by gH2AX staining is strongly recommended
The level of apoptosis from DNA-damaging conditions should be at a minimum in order to prevent apoptotic breaks from adding to the background of breakage (concomitant treatment of cells with a caspase inhibitor is encouraged)
Deficiency in DNA repair pathway(s) augments capture of DNA breaks due to their persistence in these settings
How do I know that BrITL has worked on my samples?
BrITL-Seq has been performed successfully on mouse and human cells and should be applicable to any cultured cells that proliferate and acquire persistent DNA double-strand breaks with 3' overhangs. It is recommended that gH2AX staining be performed to understand which conditions lead to optimal levels of DNA double-strand breaks in your study.
If I do not request NGS to be performed, how do I analyze my BrITL-processed samples?
Once BrITL has been performed on a sample, customers will receive an Eppendorf tube of purified DNA break fragments in 50 µL TE (retrieval) and an Eppendorf tube of initial genomic DNA in 100 µL TE (input). You can:
Perform qPCR analysis on these outputs to quantify DNA enrichment and level of breakage at specific genomic sites of interest (include a background region for comparison).
Prepare and send these samples for NGS to obtain a genome-wide view of DNA breakage.
How should I interpret my BrITL-Seq data?
BreakSight offers Bioinformatics services on customer FASTQ data (DDinsight), which calls and analyzes significant and reproducible peaks for each condition that represent DNA break sites across the genome.
Deeper analyses can include quantification of DNA repeats within and in the immediate vicinity of identified DNA break sites, as well as association studies with ENCODE or other publicly available datasets that have mapped chromatin elements (e.g. ChIP-Seq, gene regions, histone marks, etc.).