Search

Background: RNA sequencing has become the mainstay for studies of gene expression. Still, analysis of rare cells with random hexamer priming - to allow analysis of a broader range of transcripts - remains challenging., Results: We here describe a tagmentation-based, rRNA blocked, random hexamer primed RNAseq approach (T-RHEX-RNAseq) for generating stranded RNAseq libraries from very low numbers of FACS sorted cells without RNA purification steps., Conclusion: T-RHEX-RNAseq provides an easy-to-use, time efficient and automation compatible method for generating stranded RNAseq libraries from rare cells., (© 2023. The Author(s).)

Background: Chromatin immunoprecipitation coupled to sequencing (ChIP-seq) is widely used to map histone modifications and transcription factor binding on a genome-wide level., Results: We present high-throughput ChIPmentation (HT-ChIPmentation) that eliminates the need for DNA purification prior to library amplification and reduces reverse-crosslinking time from hours to minutes., Conclusions: The resulting workflow is easily established, extremely rapid, and compatible with requirements for very low numbers of FACS sorted cells, high-throughput applications and single day data generation.

Background: The use of functional genomics has largely increased our understanding of cell biology and promises to help the development of systems biology needed to understand the complex order of events that regulates cellular differentiation in vivo. One model system clearly dependent on the integration of extra and intra cellular signals is the development of B-lymphocytes from hematopoietic stem cells in the bone marrow. This developmental pathway involves several defined differentiation stages associated with specific expression of genes including surface markers that can be used for the prospective isolation of the progenitor cells directly from the bone marrow to allow for ex vivo gene expression analysis. The developmental process can be simulated in vitro making it possible to dissect information about cell/cell communication as well as to address the relevance of communication pathways in a rather direct manner. Thus we believe that B-lymphocyte development represents a useful model system to take the first steps towards systems biology investigations in the bone marrow., Results: In order to identify extra cellular signals that promote B lymphocyte development we created a database with approximately 400 receptor ligand pairs and software matching gene expression data from two cell populations to obtain information about possible communication pathways. Using this database and gene expression data from NIH3T3 cells (unable to support B cell development), OP-9 cells (strongly supportive of B cell development), pro-B and pre-B cells as well as mature peripheral B-lineage cells, we were able to identify a set of potential stage and stromal cell restricted communication pathways. Functional analysis of some of these potential ways of communication allowed us to identify BMP-4 as a potent stimulator of B-cell development in vitro. Further, the analysis suggested that there existed possibilities for progenitor B cells to send signals to the stroma. The functional consequences of this were investigated by co-culture experiments revealing that the co-incubation of stromal cells with B cell progenitors altered both the morphology and the gene expression pattern in the stromal cells., Conclusions: We believe that this gene expression data analysis method allows for the identification of functionally relevant interactions and therefore could be applied to other data sets to unravel novel communication pathways.

Background: Childhood pre-B acute lymphoblastic leukemia (ALL) is a bone marrow (BM) derived disease, which often disseminates out of the BM cavity, where malignant cells to a variable degree can be found circulating in the peripheral blood (PB). Normal pre-B cells are absolutely dependent on BM stroma for survival and differentiation. It is not known whether transformed pre-B ALL cells retain any of this dependence, which possibly could impact on drug sensitivity or MRD measurements., Results: Pre-B ALL cells, highly purified by a novel method using surface expression of CD19 and immunoglobulin light chains, from BM and PB show a very high degree of similarity in gene expression patterns, with differential expression of vascular endothelial growth factor (VEGF) as a notable exception. In addition, the cell sorting procedure revealed that in 2 out of five investigated patients, a significant fraction of the malignant cells had matured beyond the pre-B cell stage., Conclusion: The transition of ALL cells from the BM into the circulation does not demand, or result in, major changes of gene expression pattern. This might indicate an independence of BM stroma on the part of transformed pre-B cells, which contrasts with that of their normal counterparts.

Background: The expression of gene batteries, genomic units of functionally linked genes which are activated by similar sets of cis- and trans-acting regulators, has been proposed as a major determinant of cell specialization in metazoans. We developed a predictive procedure to screen the mouse and human genomes and transcriptomes for cases of gene-battery-like regulation., Results: In a screen that covered approximately 40 percent of all annotated protein-coding genes, we identified 21 co-expressed gene clusters with statistically supported sharing of cis-regulatory sequence elements. 66 predicted cases of over-represented transcription factor binding motifs were validated against the literature and fell into three categories: (i) previously described cases of gene battery-like regulation, (ii) previously unreported cases of gene battery-like regulation with some support in a limited number of genes, and (iii) predicted cases that currently lack experimental support. The novel predictions include for example Sox 17 and RFX transcription factor binding sites that were detected in approximately 10% of all testis specific genes, and HNF-1 and 4 binding sites that were detected in approximately 30% of all kidney specific genes respectively. The results are publicly available at http://www.wlab.gu.se/lindahl/genebatteries., Conclusion: 21 co-expressed gene clusters were enriched for a total of 66 shared cis-regulatory sequence elements. A majority of these predictions represent novel cases of potential co-regulation of functionally coupled proteins. Critical technical parameters were evaluated, and the results and the methods provide a valuable resource for future experimental design.