Bera, B. S. | Search bioRxiv

bioRxiv2023

Prenatal vitamin D deficiency alters immune cell proportions of young adult offspring through alteration of long-term stem cell fates

Vitamin D deficiency is a common deficiency worldwide, particularly among women of reproductive age. During pregnancy, it increases the risk of immune-related diseases in offspring later in life. However, exactly how the body remembers exposure to an adverse environment during development is poorly understood. Herein, we explore the effects of prenatal vitamin D deficiency on immune cell proportions in offspring using vitamin D deficient mice established by dietary manipulation. We found that prenatal vitamin D deficiency alters immune cell proportions in offspring by changing the transcriptional properties of genes downstream of vitamin D receptor signaling in hematopoietic stem and progenitor cells of both the fetus and adults. Our results suggest the role of cellular differentiation properties of the hematopoiesis as the long-term memories of prenatal exposure at the adult stage. Moreover, further investigations of the associations between maternal vitamin D levels and cord blood immune cell profiles from 75 healthy pregnant women and their term babies also confirm that maternal vitamin D levels in the second trimester significantly affect immune cell proportions in the babies. This highlights the importance of providing vitamin D supplementation at specific stages of pregnancy.

developmental biology↗

bioRxiv2022

An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide conjugated antibodies

BackgroundSingle-cell technologies to analyze transcription and chromatin structure have been widely used in many research areas to reveal the functions and molecular properties of cells at single-cell resolution. Sample multiplexing techniques are valuable when performing single-cell analysis, reducing technical variation and permitting cost efficiencies. Several commercially available methods are available and have been used in many scRNA-seq studies. On the other hand, while several methods have been published, the multiplexing techniques for single nuclear Assay for Transposase-Accessible Chromatin (snATAC)-seq assays remain under development. We developed a simple nucleus hashing method using oligonucleotide conjugated antibodies recognizing nuclear pore complex proteins, NuHash, to perform snATAC-seq library preparations by multiplexing. ResultsWe performed multiplexing snATAC-seq analyses on the mixture of human and mouse cell samples (two samples, 2-plex, and four samples, 4-plex) using NuHash. The demultiplexing accuracy of NuHash was high, and only ten out of 9,144 nuclei (2-plex) and 150 of 12,208 nuclei (4-plex) had discordant classifications between NuHash demultiplexing and discrimination using reference genome alignments. We compared results between snATAC-seq and deeply sequenced bulk ATAC-seq on the same samples and found that most of the peaks detected in snATAC-seq were also detected in deeply sequenced bulk ATAC-seq. The bulk ATAC-seq signal intensity was positively correlated with the number of cell subtype clusters detected in snATAC-seq, but not the subset of peaks detected in all clusters. These subsets of snATAC-seq peaks showed different distributions over different genomic features, suggesting that the peak intensities of bulk ATAC-seq can be used to identify different types of functional loci. ConclusionsOur multiplexing method using oligo-conjugated anti-nuclear pore complex proteins, NuHash, permits high accuracy demultiplexing of samples. The NuHash protocol is straightforward, it works on frozen samples, and requires no modifications for snATAC-seq library preparation.

genomics↗