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The genome-wide variant of the chromatin conformation capture technique (Hi-C) is a powerful tool for revealing patterns of genome spatial organization, as well as for understanding the effects of their disturbance on disease development. In addition, Hi-C can be used to detect chromosomal rearrangements, including balanced translocations and inversions. The use of the Hi-C method for the detection of chromosomal rearrangements is becoming more widespread. Modern high-throughput methods of genome analysis can effectively reveal point mutations and unbalanced chromosomal rearrangements. However, their sensitivity for determining translocations and inversions remains rather low. The storage of whole blood samples can affect the amount and integrity of genomic DNA, and it can distort the results of subsequent analyses if the storage was not under proper conditions. The Hi-C method is extremely demanding on the input material. The necessary condition for successfully applying Hi-C and obtaining high-quality data is the preservation of the spatial chromatin organization within the nucleus. The purpose of this study was to determine the optimal storage conditions of blood samples for subsequent Hi-C analysis. We selected 10 different conditions for blood storage and sample processing. For each condition, we prepared and sequenced Hi-C libraries. The quality of the obtained data was compared. As a result of the work, we formulated the requirements for the storage and processing of samples to obtain high-quality Hi-C data. We have established the minimum volume of blood sufficient for conducting Hi-C analysis. In addition, we have identified the most suitable methods for isolation of peripheral blood mononuclear cells and their long-term storage. The main requirement we have formulated is not to freeze whole blood.

Random transgene integration is a powerful tool for developing new genome-wide screening approaches. These techniques have already been used for functional gene annotation by transposon-insertion sequencing, for identification of transcription factor binding sites and regulatory sequences, and for dissecting chromatin position effects. Precise localization of transgenes and accurate artifact filtration are essential for this type of method. To date, many mapping assays have been developed, including Inverse-PCR, TLA, LAM-PCR, and splinkerette PCR. However, none of them is able to ensure localization of both transgene’s flanking regions simultaneously, which would be necessary for some applications. Here we proposed a cheap and simple NGS-based approach that overcomes this limitation. The developed assay requires using intentionally designed vectors that lack recognition sites of one or a set of restriction enzymes used for DNA fragmentation. By looping and sequencing these DNA fragments, we obtain special data that allows us to link the two flanking regions of the transposon. This can be useful for precise insertion mapping and for screening approaches in the field of chromosome engineering, where chromosomal recombination events between transgenes occur in a cell population. To demonstrate the method’s feasibility, we applied it for mapping SB transposon integration in the human HAP1 cell line. Our technique allowed us to efficiently localize genomic transposon integrations, which was confirmed via PCR analysis. For practical application of this approach, we proposed a set of recommendations and a normalization strategy. The developed method can be used for multiplex transgene localization and detection of rearrangements between them.

Aim. To analyze differential expression of metalloproteases genes, involved into the processes of stabilization/destabilization of atherosclerotic plaque, with the method of full genome sequencing of RNA, and to evaluate the level of metalloproteases in homogenates of plaques of various types by immune enzyme assay method (IEA).Material and methods. The study has been conducted on the specimens of atherosclerotic plaques of patients aged 45-65 y.o., inhabitants of Western Siberia with angiographically proven coronary atherosclerosis and no acute coronary syndrome, with stable angina II-IV functional class. Specimens collection from the plaques was done during an operation if there were intraoperational indications. Histology performed. In intima/media homogenates by IEA method, with BCM Diagnostics assays the levels of destructive markers were measured: MMP-1, MMP3, MMP-7, MMP-9, TIMP-1 on the Multiscan EX (Thermo Fisher Scientific, USA). Libraries preparation for full genomic sequencing of RNA was done with Illumina’s TruSeq RNA Sample Preparation Kit (Illumina, USA). Expression profile in tissues was done on HiSeq 1500 (Illumina, USA).Results. There are differences in expression of the genes MMP2, MMP7, MMP8, MMP9, MMP12, и MMP14 in different types of plaques. There was 8 times higher significant raise in increase of the expression level of ММР9 (p

Over the past few years, the CRISPR/Cas techniques have become a revolution in genome editing. Since the original paper on CRIPSR/Cas9 genome editing, researches have proposed numerous modifications of the key components of the CRISPR/Cas9 system to make it extremely efficient. Nowadays, CRISPR/Cas systems can be used not only to modify genomes, but also to control expression levels of defined genes, visualize loci of interest in the space of living cell nuclei, change methylation status of mammalian CpG sites, and to serve many other purposes. Due to an extremely high efficacy and ease of usage, the CRISPR/ Cas system has been employed in a large number of studies in various areas of biology and biotechnology. We have recently published a review describing various CRISPR/Cas systems, mechanisms of their functioning, and applications of the techniques in details. Despite the broad range of potential applications of CRISPR/Cas systems, they are mostly used for genome editing. And, however simple the system may be, there is a number of potential pitfalls on the way towards its use in CRISPR/Cas- naïve laboratory settings. In this article, we describe protocols of CRISPR/Cas9 system generation. We start with a short description of theoretical aspects underlying Cas9-mediated genome editing. Next, we describe a step-by-step protocol of guide RNA vector design and assembly, and several ways of qualitative and quantitative evaluations of the system. Finally, we report protocols of genome editing for modification of embryonic stem cells and zygotes.

The spatial organization of an eukaryotic genome plays an important role in the control of nuclear gene expression. The new Hi-C method allows investigation of the three-dimensional architecture of whole genomes. It has not been applied to study of the spatial configuration of a germ cell genome hitherto. Here we describe a protocol for production and quality control of Hi-C libraries from fibroblasts and sperm cells. Our results demonstrate that the Hi-C method can be used for studying the spatial organization of the densely packed sperm genome.

The 3D organization of an eukaryotic genome plays an important role in nuclear gene expression. Until recently, studies of this organization were limited by light microscopy and electron microscopy methods. The development of chromosome conformation capture (3С) methods allowed studying genome-wide chromosomal contacts by using only molecular methods. Nowadays, numerous 3C-based methods have been developed to reconstruct the 3D organization of eukaryotic genomes.