Morganti, R. M. | Search bioRxiv

bioRxiv2019

Mass spectrometry analysis of mouse hematopoietic stem cells and their progenitors reveals differential expression within and between proteome and transcriptome throughout adult and aged hematopoiesis

SummaryHematopoietic stem cells (HSCs) are responsible for the generation of blood and immune cells throughout life. They have the unique ability to self-renew and generate more HSCs or differentiate into a progenitor cell in response to cell-intrinsic and -extrinsic stimuli. The balance of HSC fate commitment is critical for a healthy blood supply. Imbalances during hematopoiesis, which are frequent in aging, can result in hematological malignancies and pre-malignancies as well as increase risk of atherosclerosis. Given the importance of HSCs and their progenitors, they have been extensively characterized in genomic and transcriptomic studies. However, an understanding of protein expression within the HSC compartment and more broadly throughout hematopoiesis remains poorly understood, and it has been widely reported that the correlation between mRNA and proteins is more complicated than previously thought. Previous mouse mass spectrometry studies have focused either specifically on stem and the first early progenitor or broadly across mixed populations of stem and progenitor cells, which do not allow for cell-type specific protein resolution across stages of differentiation. Mass cytometry has been employed to characterize transcription factor expression in human HSCs and progenitors but does not apply an unbiased discovery approach. New mass spectrometry technology now allows for deep proteomic coverage with no more than 200 ng of sample input. We report here a proteomics resource characterizing protein expression in mouse adult and aged HSCs, multipotent progenitors and oligopotent progenitors, 12 cell types in total. We validated differential expression by flow cytometry analysis and immunofluorescence staining. Additionally, we investigated the relationship between mRNA and protein levels of individual genes in HSCs compared to progenitors through RNA sequencing studies and identified two proteins that appear to be uniquely regulated in the HSC compartment, Cpin1 and Adnp. In summary, this resource provides proteomic coverage of adult and aged hematopoietic stem cells and their progenitors and reveals changes in protein abundance between cell types, with potential future implications in understanding mechanisms for stem-cell maintenance, niche interactions and fate determination.

cell biology↗

bioRxiv2017

Index Switching Causes "Spreading-Of-Signal" Among Multiplexed Samples In Illumina HiSeq 4000 DNA Sequencing

Illumina-based next generation sequencing (NGS) has accelerated biomedical discovery through its ability to generate thousands of gigabases of sequencing output per run at a fraction of the time and cost of conventional technologies. The process typically involves four basic steps: library preparation, cluster generation, sequencing, and data analysis. In 2015, a new chemistry of cluster generation was introduced in the newer Illumina machines (HiSeq 3000/4000/X Ten) called exclusion amplification (ExAmp), which was a fundamental shift from the earlier method of random cluster generation by bridge amplification on a non-patterned flow cell. The ExAmp chemistry, in conjunction with patterned flow cells containing nanowells at fixed locations, increases cluster density on the flow cell, thereby reducing the cost per run. It also increases sequence read quality, especially for longer read lengths (up to 150 base pairs). This advance has been widely adopted for genome sequencing because greater sequencing depth can be achieved for lower cost without compromising the quality of longer reads. We show that this promising chemistry is problematic, however, when multiplexing samples. We discovered that up to 5-10% of sequencing reads (or signals) are incorrectly assigned from a given sample to other samples in a multiplexed pool. We provide evidence that this \"spreading-of-signals\" arises from low levels of free index primers present in the pool. These index primers can prime pooled library fragments at random via complementary 3 ends, and get extended by DNA polymerase, creating a new library molecule with a new index before binding to the patterned flow cell to generate a cluster for sequencing. This causes the resulting read from that cluster to be assigned to a different sample, causing the spread of signals within multiplexed samples. We show that low levels of free index primers persist after the most common library purification procedure recommended by Illumina, and that the amount of signal spreading among samples is proportional to the level of free index primer present in the library pool. This artifact causes homogenization and misclassification of cells in single cell RNA-seq experiments. Therefore, all data generated in this way must now be carefully re-examined to ensure that \"spreading-of-signals\" has not compromised data analysis and conclusions. Re-sequencing samples using an older technology that uses conventional bridge amplification for cluster generation, or improved library cleanup strategies to remove free index primers, can minimize or eliminate this signal spreading artifact.

molecular biology↗