The ratios of the number of gene, a transcription factor known to be affected in DLBCL, while the interacting region on chromosome 14q32 lies at the immunoglobulin heavy-chain (IGH) locus (Fig
September 18, 2021
The ratios of the number of gene, a transcription factor known to be affected in DLBCL, while the interacting region on chromosome 14q32 lies at the immunoglobulin heavy-chain (IGH) locus (Fig.?5d, e), suggesting a t(3;14)(q27;q32) reciprocal translocation. we show that Low-C is highly reproducible and robust to experimental noise. To demonstrate the suitability of Low-C to analyse rare cell populations, we produce Low-C maps from primary B-cells of a diffuse large B-cell lymphoma patient. We detect a common reciprocal translocation t(3;14)(q27;q32) affecting the and IGH loci and abundant local structural variation between the patient and healthy B-cells. The ability to study chromatin conformation in primary tissue will be fundamental to fully understand the molecular pathogenesis of diseases and to eventually guide personalised therapeutic strategies. Introduction The three-dimensional (3D) organisation of chromatin in the nucleus plays a fundamental role in regulating gene expression, and its misregulation has a GW788388 major impact in developmental disorders1,2 and diseases such as cancer3. The development of chromosome conformation capture (3C)4 assays and, in particular, their recent high-throughput variants (e.g. Hi-C), have enabled the examination of 3D chromatin organisation at very high spatial resolution5,6. However, the GW788388 most widely used current experimental approaches rely on the availability of a substantial amount of starting materialon the order of millions of cellsbelow which experimental noise and low sequencing library complexity become limiting factors7. Thus far, this restricts high-resolution analyses of population Hi-C to biological questions for which large numbers of cells are available and limits the implementation of chromatin conformation analyses for rare cell populations such as those commonly obtained in clinical settings. While single-cell approaches exist8C11, they typically operate on much lower resolutions than population-based approaches and require an extensive set of specialist skills and equipment that might be out of reach for the average genomics laboratory. Recently, two methods have been developed to measure chromatin conformation using low amounts of starting material12,13. However, the lack of a systematic comparison of the data obtained with these approaches and conventional GW788388 in situ Hi-C limits our understanding of the technical constraints imposed by the amounts of starting material available. In addition, it remains to be demonstrated whether these methods could be directly applied to samples with clinical interest, such as for example, tumour samples. Here, we present Low-C, an improved in situ Hi-C method that allows the generation of high-quality genome-wide chromatin conformation maps using very low amounts of starting material. We validate this method by comparing chromatin conformation maps for a controlled cell titration, demonstrating that the obtained maps are robust down to 1,000 cells of starting material and are able to detect all conformational featurescompartments, topologically associating domains (TADs) and loopssimilarly as maps produced with a higher number of cells. Finally, we demonstrate the applicability of Low-C to clinical samples by generating chromatin conformation maps of primary B-cells from a diffuse large B-cell lymphoma (DLBCL) patient. Computational analysis of the data allows us to GW788388 detect patient-specific translocations and substantial amounts of variation in topological features. Results Low-C: A Hi-C method for low amounts of input material We first sought to develop a Hi-C method for low amounts of input GW788388 material. To do so, we modified the original in situ Hi-C protocol5, which recommends 5C10 million (M) starting cells, to allow for much smaller quantities of input material. The modifications are subtle, involving primarily changes in reagent volume and concentrations, as well as Mouse monoclonal antibody to UCHL1 / PGP9.5. The protein encoded by this gene belongs to the peptidase C12 family. This enzyme is a thiolprotease that hydrolyzes a peptide bond at the C-terminal glycine of ubiquitin. This gene isspecifically expressed in the neurons and in cells of the diffuse neuroendocrine system.Mutations in this gene may be associated with Parkinson disease timing of the individual experimental steps (Fig.?1a, Methods, Supplementary Data?1). The combined changes, however, are highly effective, allowing us to produce high-quality Hi-C libraries from starting cell numbers as low as one thousand (1?k) cells. Open in a separate window Fig. 1 Low-C enables the examination of chromatin architecture for samples with low amounts of input material. a Schematic overview of the Low-C protocol and comparison with the previously.