A single workflow for multi-species blood transcriptomics.

BACKGROUND: Blood transcriptomic analysis is widely used to provide a detailed picture of a physiological state with potential outcomes for applications in diagnostics and monitoring of the immune response to vaccines. However, multi-species transcriptomic analysis is still a challenge from a technological point of view and a standardized workflow is urgently needed to allow interspecies comparisons. RESULTS: Here, we propose a single and complete total RNA-Seq workflow to generate reliable transcriptomic data from blood samples from humans and from animals typically used in preclinical models. Blood samples from a maximum of six individuals and four different species (rabbit, non-human primate, mouse and human) were extracted and sequenced in triplicates. The workflow was evaluated using different wet-lab and dry-lab criteria, including RNA quality and quantity, the library molarity, the number of raw sequencing reads, the Phred-score quality, the GC content, the performance of ribosomal-RNA and globin depletion, the presence of residual DNA, the strandness, the percentage of coding genes, the number of genes expressed, and the presence of saturation plateau in rarefaction curves. We identified key criteria and their associated thresholds to be achieved for validating the transcriptomic workflow. In this study, we also generated an automated analysis of the transcriptomic data that streamlines the validation of the dataset generated. CONCLUSIONS: Our study has developed an end-to-end workflow that should improve the standardization and the inter-species comparison in blood transcriptomics studies. In the context of vaccines and drug development, RNA sequencing data from preclinical models can be directly compared with clinical data and used to identify potential biomarkers of value to monitor safety and efficacy.

Transcriptome-wide analysis uncovers regulatory elements of the antennal transcriptome repertoire of bumblebee at different life stages.

Bumblebees are crucial pollinators, providing essential ecosystem services and global food production. The success of pollination services relies on the interaction between sensory organs and the environment. The antenna functions as a versatile multi-sensory organ, pivotal in mediating chemosensory/olfactory information, and governs adaptive responses to environmental changes. Despite an increasing number of RNA-sequencing studies on insect antenna, comprehensive antennal transcriptome studies at the different life stages were not elucidated systematically. Here, we quantified the expression profile and dynamics of coding/microRNA genes of larval head and antennal tissues from early- and late-stage pupa to the adult of Bombus terrestris as suitable model organism among pollinators. We further performed Pearson correlation analyses on the gene expression profiles of the antennal transcriptome from larval head tissue to adult stages, exploring both positive and negative expression trends. The positively correlated coding genes were primarily enriched in sensory perception of chemical stimuli, ion transport, transmembrane transport processes and olfactory receptor activity. Negatively correlated genes were mainly enriched in organic substance biosynthesis and regulatory mechanisms underlying larval body patterning and the formation of juvenile antennal structures. As post-transcriptional regulators, miR-1000-5p, miR-13b-3p, miR-263-5p and miR-252-5p showed positive correlations, whereas miR-315-5p, miR-92b-3p, miR-137-3p, miR-11-3p and miR-10-3p exhibited negative correlations in antennal tissue. Notably, based on the inverse expression relationship, positively and negatively correlated microRNA (miRNA)-mRNA target pairs revealed that differentially expressed miRNAs predictively targeted genes involved in antennal development, shaping antennal structures and regulating antenna-specific functions. Our data serve as a foundation for understanding stage-specific antennal transcriptomes and large-scale comparative analysis of transcriptomes in different insects.

An Overview of Current Detection Methods for RNA Methylation.

Epitranscriptomic mechanisms, which constitute an important layer in post-transcriptional gene regulation, are involved in numerous cellular processes under health and disease such as stem cell development or cancer. Among various such mechanisms, RNA methylation is considered to have vital roles in eukaryotes primarily due to its dynamic and reversible nature. There are numerous RNA methylations that include, but are not limited to, 2'-O-dimethyladenosine (m6Am), N7-methylguanosine (m7G), N6-methyladenosine (m6A) and N1-methyladenosine (m1A). These biochemical modifications modulate the fate of RNA by affecting the processes such as translation, target site determination, RNA processing, polyadenylation, splicing, structure, editing and stability. Thus, it is highly important to quantitatively measure the changes in RNA methylation marks to gain insight into cellular processes under health and disease. Although there are complicating challenges in identifying certain methylation marks genome wide, various methods have been developed recently to facilitate the quantitative measurement of methylated RNAs. To this end, the detection methods for RNA methylation can be classified in five categories such as antibody-based, digestion-based, ligation-based, hybridization-based or direct RNA-based methods. In this review, we have aimed to summarize our current understanding of the detection methods for RNA methylation, highlighting their advantages and disadvantages, along with the current challenges in the field.

Efficient and cost-effective bacterial mRNA sequencing from low input samples through ribosomal RNA depletion.

BACKGROUND: RNA sequencing is a powerful approach to quantify the genome-wide distribution of mRNA molecules in a population to gain deeper understanding of cellular functions and phenotypes. However, unlike eukaryotic cells, mRNA sequencing of bacterial samples is more challenging due to the absence of a poly-A tail that typically enables efficient capture and enrichment of mRNA from the abundant rRNA molecules in a cell. Moreover, bacterial cells frequently contain 100-fold lower quantities of RNA compared to mammalian cells, which further complicates mRNA sequencing from non-cultivable and non-model bacterial species. To overcome these limitations, we report EMBR-seq (Enrichment of mRNA by Blocked rRNA), a method that efficiently depletes 5S, 16S and 23S rRNA using blocking primers to prevent their amplification. RESULTS: EMBR-seq results in 90% of the sequenced RNA molecules from an E. coli culture deriving from mRNA. We demonstrate that this increased efficiency provides a deeper view of the transcriptome without introducing technical amplification-induced biases. Moreover, compared to recent methods that employ a large array of oligonucleotides to deplete rRNA, EMBR-seq uses a single or a few oligonucleotides per rRNA, thereby making this new technology significantly more cost-effective, especially when applied to varied bacterial species. Finally, compared to existing commercial kits for bacterial rRNA depletion, we show that EMBR-seq can be used to successfully quantify the transcriptome from more than 500-fold lower starting total RNA. CONCLUSIONS: EMBR-seq provides an efficient and cost-effective approach to quantify global gene expression profiles from low input bacterial samples.

Total RNA and genomic DNA of Lactobacillus gasseri OLL2809 induce interleukin-12 production in the mouse macrophage cell line J774.1 via toll-like receptors 7 and 9.

BACKGROUND: Lactobacillus gasseri OLL2809 can highly induce interleukin (IL)-12 production in immune cells. Even though beneficial properties of this strain for both humans and animals have been reported, the mechanism by which the bacteria induces the production of IL-12 in immune cells remains elusive. In this study, we investigated the mechanism of induction of IL-12 using a mouse macrophage cell line J774.1. RESULTS: Inhibition of phagocytosis of L. gasseri OLL2809, and myeloid differentiation factor 88 and Toll-like receptors (TLRs) 7 and 9 signalling attenuated IL-12 production in J774.1 cells. Total RNA and genomic DNA of L. gasseri OLL2809, when transferred to the J774.1 cells, also induced IL-12 production. The difference in the IL-12-inducing activity of Lactobacilli is attributed to the susceptibility to phagocytosis, but not to a difference in the total RNA and genomic DNA of each strain. CONCLUSION: We concluded that total RNA and genomic DNA of phagocytosed L. gasseri OLL2809 induce IL-12 production in J774.1 cell via TLRs 7 and 9, and the high IL-12-inducing activity of L. gasseri OLL2809 is due to its greater susceptibility to phagocytosis.

Profiling of RNAs from Human Islet-Derived Exosomes in a Model of Type 1 Diabetes.

Type 1 diabetes (T1D) is characterized by the immune-mediated destruction of insulin-producing islet ß cells. Biomarkers capable of identifying T1D risk and dissecting disease-related heterogeneity represent an unmet clinical need. Toward the goal of informing T1D biomarker strategies, we profiled coding and noncoding RNAs in human islet-derived exosomes and identified RNAs that were differentially expressed under proinflammatory cytokine stress conditions. Human pancreatic islets were obtained from cadaveric donors and treated with/without IL-1ß and IFN-γ. Total RNA and small RNA sequencing were performed from islet-derived exosomes to identify mRNAs, long noncoding RNAs, and small noncoding RNAs. RNAs with a fold change ≥1.3 and a p-value <0.05 were considered as differentially expressed. mRNAs and miRNAs represented the most abundant long and small RNA species, respectively. Each of the RNA species showed altered expression patterns with cytokine treatment, and differentially expressed RNAs were predicted to be involved in insulin secretion, calcium signaling, necrosis, and apoptosis. Taken together, our data identify RNAs that are dysregulated under cytokine stress in human islet-derived exosomes, providing a comprehensive catalog of protein coding and noncoding RNAs that may serve as potential circulating biomarkers in T1D.

Low-cell-number, single-tube amplification (STA) of total RNA revealed transcriptome changes from pluripotency to endothelium.

BACKGROUND: In addition to messenger RNA (mRNA), noncoding RNAs (ncRNAs) are essential components in cellular machineries for translation and splicing. Besides their housekeeping functions, ncRNAs are involved in cell type-specific regulation of translation, mRNA stability, genome structure, and accessibility. To have a comprehensive understanding of the identities and functions of different cell types, a method to comprehensively quantify both mRNA and ncRNA in a sensitive manner is highly desirable. METHODS: Here we tried to develop a system capable of concurrently profiling both mRNA and ncRNA by polyadenylating RNA in samples before reverse transcription. The sensitivity of the system was maximized by avoiding purification from cell lysis to amplified cDNA and by optimizing the buffer conditions. The single-tube amplification (STA) system was applied to single to 100 cells of 293T cells, human pluripotent stem cells (hPSCs) and their differentiated endothelial progenies to validate its quantitative power and sensitivity by qPCR and high-throughput sequencing. RESULTS: Using microRNA (miRNA) as an example, we showed that complementary DNA (cDNA) from ncRNAs could be amplified and specifically detected from a few cells within a single tube. The sensitivity of the system was maximized by avoiding purification from cell lysis to amplified cDNA and by optimizing the buffer conditions. With 100 human embryonic stem cells (hESCs) and their differentiated endothelial cells as input for high-throughput sequencing, the single-tube amplification (STA) system revealed both well-known and other miRNAs selectively enriched in each cell type. The selective enrichment of the miRNAs was further verified by qPCR with 293FT cells and a human induced pluripotent stem cell (hiPSC) line. In addition, the detection of other non-miRNA transcripts indicated that the STA target was not limited to miRNA, but extended to other ncRNAs and mRNAs as well. Finally, the STA system was capable of detecting miRNA and mRNA expression down to single cells, albeit with some loss of sensitivity and power. CONCLUSIONS: Overall, STA offered a simple and sensitive way to concurrently quantify both mRNA and ncRNA expression in low-cell-number samples for both qPCR and high-throughput sequencing.

Analysis of Different Approaches for the Selection of Reference Genes in RT-qPCR Experiments: A Case Study in Skeletal Muscle of Growing Mice.

The reliability of reverse transcription-quantitative PCR (RT-qPCR) results in gene expression studies depends on the approaches used to account for non-biological variations. In order to find a proper normalization strategy for the study of genes related to growth hormone signaling in skeletal muscle of growing mice, nine unrelated genes were evaluated as internal controls. According to the most used algorithms-geNorm, the Comparative ΔCq method, NormFinder and BestKeeper-GSK3B, YWHAZ, RPL13A and RN18S were found as the most stable. However, the relative expression levels of eight of the potential reference genes assessed decreased with age in cDNA samples obtained from the same amount of total RNA. In a different approach to analyze this apparent discrepancy, experiments were performed with cDNA obtained from equal amounts of purified mRNA. Since the decline was still observed, the hypothesis of an age-related change in mRNA to total RNA ratio that could account for the systematic decrease was rejected. Differences among experimental groups could be due to a substantial increase with age in highly expressed mRNAs, which would bias the quantitation of the remaining genes. Consequently, those reference genes reflecting this dilution effect, which would have been discarded considering their variable relative expression levels, arose as suitable internal controls.

[Systemic delivery of complexes of melanoma RNA with mannosylated liposomes activates highly efficient murine melanoma-specific cytotoxic T cells in vivo].

The efficiency of the antitumor immune response triggered by dendritic cell (DC)-based vaccines depends predominantly on the efficiency of delivering tumor antigen-coding nucleic acids into DCs. Mannosylated liposomes were used to deliver tumor total RNA into DCs both ex vivo and in vivo, and the cytotoxic T-lymphocyte (CTL) antitumor response was assayed. The liposomes contained the mannosylated lipid conjugate 3-[6-(α-D-mannopyranosyloxy)hexyl]amino-4-{6-[rac-2,3-di(tetradecyloxy)prop-1-yl oxycarbonylamino]hexyl}aminocyclobut-3-en-1,2-dione), the polycationic lipid 2X3 (1,26-bis(cholest-5-en-3ß-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride), and the zwitterionic lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) at a molar ratio of 1: 3: 6 and were used as a transfection agent. Total RNA isolated from B16-F10 mouse melanoma cells served as a source of tumor antigens. Systemic administration of mannosylated liposomes-tumor RNA complexes into circulation of melanoma-bearing mice induced an efficient CTL response, which reduced the melanoma cell index in vitro with the same efficiency (by a factor of 2.8) as CTLs activated via an inoculation of DCs loaded with complexes of the same composition ex vivo. Complexes of tumor RNA with control liposomes, which lacked the mannosylated lipid conjugate, or DCs transfected with these complexes ex vivo were less efficient and reduced the melanoma cell count by a factor of only 1.6-1.8.

A new method for stranded whole transcriptome RNA-seq.

This report describes an improved protocol to generate stranded, barcoded RNA-seq libraries to capture the whole transcriptome. By optimizing the use of duplex specific nuclease (DSN) to remove ribosomal RNA reads from stranded barcoded libraries, we demonstrate improved efficiency of multiplexed next generation sequencing (NGS). This approach detects expression profiles of all RNA types, including miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (long non-coding RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA) without the use of gel electrophoresis. The improved protocol generates high quality data that can be used to identify differential expression in known and novel coding and non-coding transcripts, splice variants, mitochondrial genes and SNPs (single nucleotide polymorphisms).

Comparison of RNA isolation methods from insect larvae.

Isolating RNA from insects is becoming increasingly important in molecular entomology. Four methods including three commercial kits RNeasy Mini Kit (Qiagen), SV Total RNA isolation system (Promega), TRIzol reagent (Invitrogen), and a cetyl trimethylammonium bromide (CTAB)-based method were compared regarding their ability to isolate RNA from whole-body larvae of Thaumatotibia leucotreta (Meyrick), Thanatophilus micans (F.), Plutella xylostella (L.), and Tenebrio molitor (L.). A difference was observed among the four methods regarding RNA quality but not quantity. However, RNA quality and quantity obtained was not dependent on the insect species. The CTAB-based method produced low-quality RNA and the Trizol reagent produced partially degraded RNA, whereas the RNeasy Mini Kit and SV Total RNA isolation system produced RNA of consistently high quality. However, after reverse transcription to cDNA, RNA produced using all four extraction methods could be used to successfully amplify a 708 bp fragment of the cytochrome oxidase I gene. Of the four methods, the SV Total RNA isolation system showed the least amount of DNA contamination with the highest RNA integrity number and is thus recommended for stringent applications where high-quality RNA is required. This is the first comparison of RNA isolation methods among different insect species and the first to compare RNA isolation methods in insects in the last 20 years.

Comparison of Fecal MicroRNA Isolation Using Various Total RNA Isolation Kits.

Fecal specimens have long been regarded as promising sources for gastrointestinal cancer screening and have, thus, been extensively investigated in biomarker research. MicroRNAs (miRNAs) are small, non-coding RNA molecules involved in regulating various biological processes. They are commonly dysregulated during tumor development and exhibit differential expression in feces. To assess the preanalytical feasibility of fecal miRNA analysis, we systematically compared the performance of commonly used total RNA extraction methods. Fecal samples from healthy subjects were utilized for this evaluation. Various methods, including miRNeasy, Universal, Trizol, RNeasy, and mirVana kits, were employed to isolate total RNA. MiRNA expression analyses were conducted using TaqMan or SYBR Green qRT-PCR for a subset of miRNAs, with externally spiked-in cel-miR-39 used for normalization. Most methods demonstrated similar performance in terms of the total RNA concentration and purity. Externally spiked cel-miR-39 and endogenous miRNAs (RNU6b, miR-16, and miR-21) exhibited comparable concentrations across the different RNA isolation methods, whereas the RNeasy mini kit consistently yielded lower values. Our findings suggest that various isolation methods produce reproducible and comparable miRNA expression results, supporting the potential comparability and translational applicability of miRNA-based biomarker research in the future.

Evaluation of SARS-CoV-2 quantification from oropharyngeal swabs, nasopharyngeal swabs, and naso-oropharyngeal swabs: A cross-sectional study.

The current naso-oropharyngeal swab for SARS-CoV-2 detection faces several problems, such as waste issues and its use for quantitative studies. This study aimed to evaluate the total RNA and viral loads from different upper respiratory tract swabs types and whether SARS-CoV-2 quantification can use the current internal control for normalization. This cross-sectional study collected positive specimens with single oropharyngeal or nasopharyngeal swabs and naso-oropharyngeal swabs. The samples were extracted, tested with qualitative RT‒PCR, and then tested with quantitative RT‒PCR. The RNA eluate was measured for the total RNA concentration. The total RNA concentration, viral load, and RNaseP Ct values were collected and analysed statistically. The positive results came from 41 oropharyngeal swabs, 34 nasopharyngeal swabs, and 36 naso-oropharyngeal swabs. The total RNA increased significantly from oropharyngeal swabs to nasopharyngeal swabs to naso-oropharyngeal swabs. Significant differences in RNaseP Ct values between groups and their correlations with total RNA were found. In addition, the increase in the total RNA and the RNaseP Ct values were unrelated to the viral load. The physical features in the naso-oropharyngeal area and the swabbing procedures could affect the total RNA but not the viral load. However, since the virus particles could present inside and outside human cells, the increase in collected human cells may not always be followed by the viral load increase. Normalization using the RNaseP Ct value became unnecessary due to the factors mentioned above. Therefore, a careful approach is needed in viral load studies of swab specimens.

Improved heat shock method for extracting total RNA from nasopharyngeal swab samples even with low viral load.

This study aimed to improve the heat shock method as a cost-effective and time-efficient for total RNA extraction. We compared the effectiveness of two total RNA extraction methods by using Real-Time PCR for nasopharynx swabs. Include: I; use of a commercial total RNA extraction kit as a standard. II; utilized a modified heat shock method (MHS). Time, centrifuge speed and duration, proteinase K, and RNA carrier were optimized. The optimized parameters included treating the sample with 5 µg/µL at 56°C for 5 minutes, heating at 95°C for 5 minutes followed by thermal shock in ice for 3 minutes, adding 4 µg/µL RNA carrier at room temperature for 3 minutes, and centrifuging at 7000 rpm for 10 minutes. This optimization demonstrated a sensitivity and specificity of 100% (CI: 95%) even in samples with low viral load. Our in-house method presents a rapid, and cost-effective alternative for total RNA extraction.

Pan-Cancer Single-Nucleus Total RNA Sequencing Using snHH-Seq.

Tumor heterogeneity and its drivers impair tumor progression and cancer therapy. Single-cell RNA sequencing is used to investigate the heterogeneity of tumor ecosystems. However, most methods of scRNA-seq amplify the termini of polyadenylated transcripts, making it challenging to perform total RNA analysis and somatic mutation analysis.Therefore, a high-throughput and high-sensitivity method called snHH-seq is developed, which combines random primers and a preindex strategy in the droplet microfluidic platform. This innovative method allows for the detection of total RNA in single nuclei from clinically frozen samples. A robust pipeline to facilitate the analysis of full-length RNA-seq data is also established. snHH-seq is applied to more than 730 000 single nuclei from 32 patients with various tumor types. The pan-cancer study enables it to comprehensively profile data on the tumor transcriptome, including expression levels, mutations, splicing patterns, clone dynamics, etc. New malignant cell subclusters and exploring their specific function across cancers are identified. Furthermore, the malignant status of epithelial cells is investigated among different cancer types with respect to mutation and splicing patterns. The ability to detect full-length RNA at the single-nucleus level provides a powerful tool for studying complex biological systems and has broad implications for understanding tumor pathology.

Sexually dimorphic DNA methylation and gene expression patterns in human first trimester placenta.

BACKGROUND: Fetal sex and placental development impact pregnancy outcomes and fetal-maternal health, but the critical timepoint of placenta establishment in first trimester is understudied in human pregnancies. METHODS: Pregnant subjects were recruited in late first trimester (weeks 10-14) at time of chorionic villus sampling, a prenatal diagnostic test. Leftover placenta tissue was collected and stored until birth outcomes were known, then DNA and RNA were isolated from singleton, normal karyotype pregnancies resulting in live births. DNA methylation was measured with the Illumina Infinium MethylationEPIC BeadChip array (n = 56). Differential methylation analysis compared 25 females versus 31 males using a generalized linear model on 743,461 autosomal probes. Gene expression sex differences were analyzed with RNA-sequencing (n = 74). An integrated analysis was performed using linear regression to correlate gene expression and DNA methylation in 51 overlapping placentas. RESULTS: Methylation analysis identified 151 differentially methylated probes (DMPs) significant at false discovery rate < 0.05, including 89 (59%) hypermethylated in females. Probe cg17612569 (GABPA, ATP5J) was the most significant CpG site, hypermethylated in males. There were 11 differentially methylated regions affected by fetal sex, with transcription factors ZNF300 and ZNF311 most significantly hypermethylated in males and females, respectively. RNA-sequencing identified 152 genes significantly sexually dimorphic at false discovery rate < 0.05. The 151 DMPs were associated with 18 genes with gene downregulation (P < 0.05) in the direction of hypermethylation, including 2 genes significant at false discovery rate < 0.05 (ZNF300 and CUB and Sushi multiple domains 1, CSMD1). Both genes, as well as Family With Sequence Similarity 228 Member A (FAM228A), showed significant correlation between DNA methylation and sexually dimorphic gene expression, though FAM228A DNA methylation was less sexually dimorphic. Comparison with other sex differences studies found that cg17612569 is male-hypermethylated across gestation in placenta and in human blood up to adulthood. CONCLUSIONS: Overall, sex dimorphic differential methylation with associated differential gene expression in the first trimester placenta is small, but there remain significant genes that may be regulated through methylation leading to differences in the first trimester placenta.

Fetal sex and placenta development affect pregnancy outcomes for both the fetus and mother throughout pregnancy, including risk of miscarriages, preterm birth, preeclampsia, and other outcomes. Epigenetics, the "overlay" of regulatory signals on DNA which affects how DNA is read, is not well understood in early pregnancy when critical placenta developments are happening that affect the rest of pregnancy. Here, we use leftover placenta biopsy samples (n = 56) donated by Cedars-Sinai patients with informed consent to learn about first trimester human placenta DNA methylation differences due to fetal sex. Out of the total 743,461 sites analyzed, we identified 151 sites significantly affected by fetal sex after correcting p-values to reduce false positives (false discovery rate < 0.05). We also performed an analysis to look at multiple sites and identified 11 regions across the genome with significant DNA methylation changes due to fetal sex. Furthermore, because DNA methylation is a regulatory mark on DNA which typically dampens gene expression, we also compared the DNA methylation sex differences to placental RNA-sequencing gene expression analysis using the same tissue from a mostly overlapping patient group (n = 74 total sequenced, n = 51 overlap). We identify 18 genes which show both significant DNA methylation differences and gene expression changes. The most significant gene was transcription factor ZNF300 with higher DNA methylation in males and reduced gene expression in males (and thus higher gene expression in females). This study identifies some sex differences that continue until later pregnancy and others that are unique to first trimester.

High-quality total RNA extraction from early-stage lamprey embryos.

High-purity total RNA extraction from animal embryos is essential for transcriptome analyses. lampreys, together with hagfish, are the only extant jawless vertebrates or cyclostomes and are thus key organisms for EvoDevo studies. However, extracting uncontaminated RNA from early-stage embryos remains challenging. RNA does not bind to the silica membrane in filter-based extractions, significantly reducing yields; and ethanol/isopropanol precipitation methods lead to contaminants, bringing down the optical density (OD) 260/280 ratio. The RNA extraction protocol was modified using precentrifugation and adding salts before isopropanol precipitation. This modification significantly increased RNA yield, removed contaminants and improved RNA integrity. Egg membrane sources were suspected to cause RNA purification problems because low-quality extraction does not occur in posthatching embryos.

Approaches for single-cell RNA sequencing across tissues and cell types.

Single-cell sequencing of RNA (scRNA-seq) has advanced our understanding of cellular heterogeneity and signaling in developmental biology and disease. A large number of complementary assays have been developed to profile transcriptomes of individual cells, also in combination with other readouts, such as chromatin accessibility or antibody-based analysis of protein surface markers. As scRNA-seq technologies are advancing fast, it is challenging to establish robust workflows and up-to-date protocols that are best suited to address the large range of research questions. Here, we review scRNA-seq techniques from mRNA end-counting to total RNA in relation to their specific features and outline the necessary sample preparation steps and quality control measures. Based on our experience in dealing with the continuously growing portfolio from the perspective of a central single-cell facility, we aim to provide guidance on how workflows can be best automatized and share our experience in coping with the continuous expansion of scRNA-seq techniques.

Comprehensive Analysis and Drug Modulation of Human Endogenous Retrovirus in Hepatocellular Carcinomas.

BACKGROUND: Human endogenous retroviruses (HERVs) play an important role in the development of cancer and many diseases. Here, we comprehensively explored the impact of HERVs on hepatocellular carcinomas (HCCs). METHODS: We employed Telescope to identify HERVs and quantify their expression in the total RNA sequencing data obtained from 254 HCC samples, comprising 254 tumor tissues and 34 matched normal tissues. RESULTS: In total, 3357 locus-specific activations of HERVs were differentially expressed, and 180 were correlated with patient survival. Using these 180 HERVs for classification, we found four subgroups with survival correlation. Higher expression levels of the 180 HERVs were correlated with poorer survival, while age, AFP, some mutations, and copy and structural variants differed among subgroups. The differential expression of host genes in high expression of these 180 HERVs primarily involved the activation of pathways related to immunity and infection, lipid and atherosclerosis, MAPK and NF-kB signaling, and cytokine-cytokine receptor interactions. Conversely, there was a suppression of pathways associated with RNA processing, including nucleocytoplasmic transport, surveillance and ribosome biogenesis, and transcriptional misregulation in cancer pathways. Almost all genes involved in HERV activation restriction, KRAB zinc finger proteins, RNA nucleocytoplasmic transport, stemness, HLA and antigen processing and presentation, and immune checkpoints were overexpressed in cancerous tissues, and many over-expressed HERV-related nearby genes were correlated with high HERV activation and poor survival. Twenty-three immune and stromal cells showed higher expression in non-cancerous than cancerous tissues, and seven were correlated with HERV activation. Small-molecule modulation of alternative splicing (AS) altered the expression of survival-related HERVs and their activation-related genes, as well as nearby genes. CONCLUSION: Comprehensive and integrated approaches for evaluating HERV expression and their correlation with specific pathways have the potential to provide new companion diagnostics and therapeutic strategies for HCC.

Diversity and composition of active and total bacteria in rhizospheric soil in response to continuous cropping years of Panax notoginseng.

The synchronous research and analysis of total and active soil microbial communities can provide insight into how these communities are impacted by continuous cropping years and pathogen infection. The diversity of total and active bacteria in rhizospheric soil of 2-year-old and 3-year-old healthy and diseased Panax notoginseng can comprehensively reveal the bacterial response characteristics in continuous cropping practice. The results showed that 4916 operational taxonomic units (OTUs) were found in the rhizospheric soil bacterial community of P. notoginseng at the DNA level, but only 2773 OTUs were found at the RNA level. The rhizospheric environment had significant effects on the active and bacterial communities, as indicated by the number of OTUs, Shannon, Chao1, Faith's phylogenetic diversity (Faith's PD), and Simpson's diversity indexes. The DNA level can better show the difference in diversity level before and after infection with root rot. The bacterial Chao1 and Faith's PD diversity indexes of 2-year-old root rot-diseased P. notoginseng rhizospheric soil (D2) were higher than that of healthy plants, while the bacterial Shannon diversity index of 3-year-old root rot-diseased P. notoginseng rhizospheric soil (D3) was the lowest in the total bacteria. Principal coordinate analysis (PCoA) illustrated that the total bacterial species composition changed markedly after root rot disease. There were significant differences in the composition of active bacterial species between the 2-year and 3-year rhizospheres. In conclusion, the total and active edaphic rhizospheric bacterial communities could provide important opportunities to understand the responses of bacteria to continuous cropping of P. notoginseng. Differential responses of total and active edaphic rhizosphere bacterial communities to continuous cropping of Panax notoginseng.