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Longest transcripts without isoforms

Longest transcripts without isoforms


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Does anyone know what genes produce the longest mRNAs? I am planning a project on synthetic introns and want to use stuffer fragments to vary intron size. Obviously they must not contain any splicing sites, hence I plan on buying cDNA clones.

It would be incredibly appreciated if someone could point fingers at transcripts that are 12… 20 kbp long (and ideally have no other known isoforms or cryptic splicing sites).

Thank you!


To R!

  1. Download the most comprehensive GTF file from Gencode for mouse.
  2. Do the following in R (I'll put comments in the code so you can hopefully follow along):
library(GenomicFeatures) #Load the GTF file and make a TxDb object txdb = makeTxDbFromGFF("gencode.vM13.annotation.gtf", format="gtf") #Make a GRangesList, with transcripts split per gene grl = transcriptsBy(txdb, by="gene") # Filter the GRangesList for genes with one annotated isoform grl2 = grl[which(elementNROWS(grl) == 1)] # Make a new GRangesList of the exons per gene from above grl = exonsBy(txdb, by="gene") grl = grl[which(names(grl) %in% singleIsoformGenes)] # Get the length of each single-isoform gene lens = sum(width(grl)) # Get the top 10 single isoform genes by length head(lens[order(lens, decreasing=T)], n=10)

You then get the following output, with gene IDs on top and lengths below that:

ENSMUSG00000020255.8 ENSMUSG00000101609.1 ENSMUSG00000104211.1 123179 84395 74456 ENSMUSG00000109536.1 ENSMUSG00000109125.1 ENSMUSG00000047888.9 30942 25241 17327 ENSMUSG00000022262.7 ENSMUSG00000066108.7 ENSMUSG00000033826.9 15630 14964 14583 ENSMUSG00000032855.5 14170

NCBI RefSeq Select

The RefSeq Select dataset consists of a representative or “Select” transcript for every protein-coding gene. The transcript is chosen by an automated pipeline based on multiple selection criteria, which include prior use in clinical databases (e.g., Locus Reference Genomic), transcript expression, conservation of the coding region, transcript and protein length and concordance with the Swiss-Prot canonical isoform. The RefSeq Select transcript is usually well-supported by archived data, well-expressed, conserved and represents the biology of the gene.


Background

Plants undergo constant exposure to highly variable environmental stresses during their life cycles, with salinity stress representing the leading constraint to growth and productivity, which is responsible for quality and yield [1]. In general, salinity interferes with plant growth because it imposes two main stresses on plants: hyperosmotic pressure, resulting from the low water availability, and ion toxicity (mainly Na + ), arising from solute imbalances [2]. For plants to survive under this stress condition, they will employ intricate defense mechanisms through a series of drastic physiological and biochemical changes [3]. These modifications include maintaining the integrity of the cell membrane, regulating water balance, scavenging reactive oxygen species (ROS), and accumulating compatible solutes, as well as reinstating cellular ionic equilibrium [4], which are all dedicated to reducing the osmotic or ionic damage caused by salinity.

The physiological responses of plants acclimating to unfavourable environments are all initiated upon the activation of cascades of molecular networks within the signalling pathways [5]. In the signalling pathways, high salinity level often triggers an increase in cytosolic Ca 2+ , ROS, and ABA, which are critical signal transduction components [6]. Activated Ca 2+ , ROS, and ABA signalling cascades further alter plant transcriptomes by regulating downstream transcription factors (TFs), such as AP2-EREBPs, MYBs, and bHLHs. Thereafter, these TFs can cause changes in the expression of various osmotic stress-responsive genes, such as P5CSs and COR15As, and ionic stress-responsive genes, such as NHXs and HKTs, which ultimately contribute to plant salinity tolerance [7, 8]. Recently, several well characterized signalling pathways of plants responding to salinity stress have been revealed, such as the calcium-dependent protein kinase (CDPK) pathway, which plays a critical role in osmotic stress response [9] the salt overly sensitive (SOS) pathway, which is activated by Ca 2+ spikes from the cytoplasm and overcomes ionic damage by maintaining cellular ion homeostasis [10] and the calcineurin B-like proteins–CBL-interacting protein kinases (CBL-CIPK) module, which is essential to combating both osmotic and ionic stress [11]. Despite the progress that has been made in detailing these processes, the underlying mechanisms of plants’ response to salinity need further exploration, especially in non-model plants.

Alfalfa (Medicago sativa L.), is the most widely cultivated perennial forage legume, and more than 40 million hectares are planted worldwide [12]. This species is referred to as the “queen of forages” and is used as hay, silage, and pasture for ruminants and dairy production [13]. Moreover, alfalfa possesses considerable potential as a biofuel feedstock for ethanol production [14]. In China, alfalfa plantation areas are mainly distributed in the northern, northwestern, and northeastern regions [15]. Unfortunately, soil salinization is dramatically increasing in those areas, which dramatically limits the productivity and persistence of alfalfa [16]. Therefore, it is imperative to perform studies on the molecular mechanisms of adaptation to salinity stress in alfalfa.

Previous studies have shown that overexpression of the stress-associated genes encoding the compatible solute AgcodA [17], ion transporter SeNHX1 [18], protein kinase AtNDPK2 [19], or TFs GmDREB1 [20] and GsWRKY20 [21] results in enhanced tolerance to salinity stress in alfalfa. Given the relatively low-throughput characteristics of genetic-based approaches, large-scale potential genes involved in alfalfa responses to adverse salinity stimuli have been studied via next-generation sequencing (NGS) technologies. Postnikova et al. (2013) performed the transcriptional profiling of alfalfa whole roots under NaCl stress for 7 days in two distinct salinity-tolerant germplasms their results showed that salinity-responsive genes are mainly involved in stabilization of the plasma membrane and several salinity-responsive TF families [22]. Lei et al. (2018) comparatively analyzed the leaf transcriptomes under NaCl stress for 7 days between two different salinity-tolerant alfalfa cultivars, which revealed that plant hormone interactions is a vital regulator in alfalfa to maintain specific physiological status for adaptation to salinity stress [23]. Furthermore, a de novo transcriptional analysis of whole alfalfa seedlings treated with saline–alkaline solutions for 0, 1, and 7 days indicated that antioxidant capacity was one of the central mechanisms underlying alfalfa’s saline–alkaline stress tolerance [24]. However, these studies mainly focused on genotype-specific salinity tolerance mechanisms or more complex saline–alkaline tolerance mechanisms, systematic consensus on the comparative damage caused by osmotic versus ionic stresses when alfalfa is subjected to salinity is still lacking. And also, even with these transcriptional-based NGS methods, the disadvantage was clear, such as the short lengths of sequencing reads, which greatly hinders its ability to estimate transcript abundance at genome-wide scale.

Luckily, the PacBio RSII third-generation sequencing technology can overcome these limitations. Compared with traditional NGS technologies, this technology accomplishes single molecule real-time (SMRT) isoform sequencing (Iso-Seq) with long read lengths, uniform coverage, and high accuracy, which renders PacBio RSII very effective at capturing the full catalogue of transcripts and constructs of a comprehensive transcriptome for species without genome sequence [25]. Furthermore, RNA-Seq based on the BGISEQ-500 platform has been applied to gene expression comparisons of different species, developmental stages, and stresses [26, 27]. Currently, to our knowledge, genome-wide transcriptomic analysis of the salinity-responsive genes has not been reported in alfalfa root tips, where is the primary site for the perception of hyperosmotic pressure and ion toxicity [28, 29]. Thus, for the first time, we applied the Iso-Seq protocol to generate a full-length reference transcriptome for alfalfa root tips during continuous NaCl (an iso-osmotic stressor) and mannitol (a non-ionic osmotic stressor) treatments and then performed a gene expression comparison for the same stressed samples at the transcriptional scale using BGISEQ-500 RNA-Seq. Moreover, the physiological effects of NaCl and mannitol treatments on ROS accumulation and cell damage, as well as underlying antioxidant and osmoprotectant responses, were determined. The results of this study will help us to understand the contribution of the two osmotic and ionic components towards salinity tolerance in alfalfa.


Introduction

Analysis of transcriptomes, which represent the activity of genes in the genome, is vital for understanding the relationship between genotype and phenotype. The dynamics and complexity of transcriptome regulate all aspects of plant growth, development, and responses to various external biotic and abiotic cues. Different methods such as expressed sequence tag (EST) sequencing (Wu et al., 2002), serial analysis of gene expression (SAGE) (Matsumura et al., 1999), DNA microarray (Hihara et al., 2001), and recently RNA sequencing (RNA-Seq) using next-generation sequencing (NGS) technologies (Mortazavi et al., 2008) have been developed to analyze transcriptomes. Since 2005, second-generation short-read sequencing platforms quickly replaced first-generation Sanger sequencing technology for various high-throughput applications due to lower costs and greater sequencing depth (Sedlazeck et al., 2018). However, the read length is the major limitation in second-generation short-read sequencing, which made it harder to analyze several aspects of co/post-transcriptional processing events. To overcome this limitation, in the past few years, researchers are sequencing full-length transcripts mostly using two platforms, Pacific BioSciences (PacBio) (Rhoads and Au, 2015) and Oxford Nanopore Technologies (ONT) (Bayega et al., 2018), which are referred to as “third” and 𠇏ourth” generation sequencing technologies, respectively (Slatko et al., 2018). These two platforms increased read length considerably as compared to other NGS methods and can, therefore, be used to address a larger variety of research questions. Single-molecule real-time (SMRT) isoform sequencing (Iso-Seq) using PacBio platform captures the full length of transcripts (Gonzalez-Garay, 2016) and thereby presents easier and more accurate ways for different applications, such as gene annotation (Zhao et al., 2018), isoform identification (Abdel-Ghany et al., 2016 Wang T. et al., 2017), identification of fusion transcripts (Weirather et al., 2015), and long non-coding RNA (lncRNA) discovery (Li et al., 2016). Here, we discuss applications and broader utility of PacBio and ONT in transcriptome studies. Recently developed direct RNA-Seq using nanopore can avoid amplification biases (Garalde et al., 2018). Furthermore, this technology has the potential to provide a complete view of RNA modifications such as N 6 -methyladenosine, 5-methylcytidine, and 5-hydroxylmethylcytidine (Li X. et al., 2017), which are collectively referred to as the 𠇎pitranscriptome.”

Parts of the core algorithm for PacBio and ONT long-read analyses are similar to short-read analysis strategies used in second-generation sequencing approaches. Nevertheless, specific new bioinformatics tools have been designed for several of the applications, which have not been part of second-generation sequencing pipelines. These tools are needed to provide greater flexibility to achieve different goals as well as to address new issues, such as higher error rates and low throughput. We present currently available bioinformatics methods for PacBio and ONT read analysis, including reads-of-interest (ROI) extraction, error correction (Au et al., 2012), mapping (Wu and Watanabe, 2005), isoform clustering (Fu et al., 2012), and identification of multiple transcript isoforms (Abdel-Ghany et al., 2016). Improvements in these new methods and computational pipelines will expand the landscape of transcriptome complexity at the transcript isoform and epitranscriptome level with higher throughput and higher accuracy. Here, we discussed PacBio Iso-Seq and ONT direct RNA-Seq methodologies, the current status of bioinformatics tools used to analyze the long-reads and highlighted various applications of these methods.


Discussion

Here we show widespread shortening of 3’UTR by APA in spermatogenesis, with 3’UTRs being the shortest in spermatids. While this mechanism appears to be general, some genes tend to be regulated to a greater extent than others. Testis-specific genes display more substantial 3’UTR shortening than ubiquitously expressed genes, highlighting the importance of this mechanism for spermatogenesis. Interestingly, protein ubiquitination is the most significant pathway associated with genes with 3’UTR shortening. Because of the importance of ubiquitination for sperm development after spermatids, such as nucleosome removal and remodeling of cell structure, we posit that 3’UTR shortening plays an important role for spermiogenesis. Further experimental studies are needed to confirm this hypothesis.

Using genes without APA as a control group, we reveal that 3’UTR elements play an important role in mRNA abundance during spermatogenesis, including U-rich, UA-rich and UG-rich elements, possibly through regulation of mRNA stability. This may be important for elimination of mRNAs before spermatozoa, which contain little mRNA [61]. Future studies are needed to elucidate proteins involved in the mRNA degradation mediated by these cis elements. Another important question to be addressed is how much of a role APA plays to help transcripts evade degradation. Compared to sUTR transcripts, long 3’UTR isoforms decreased in expression by

1.7-fold from 2 weeks to 4 weeks and short isoforms increased by about 2.1-fold in the same period (Additional file 1: Figure S7). Assuming long isoforms have similar decay rates to sUTR transcripts, their decrease in expression can be attributable to APA changes. Therefore, while future experiments are needed to precisely address the question, our analysis indicates a significant impact on mRNA stability through APA changes. In the same vein, our cis element analysis result can parsimoniously explain why some genes display 3’UTR lengthening while the global trend is shortening: for genes whose cUTRs are enriched with destabilizing elements, their short 3’UTR isoforms are less stable than the long isoform, which presumably contains additional stabilizing elements in the aUTR to negate the destabilizing effect of cUTR, leading to overall display of 3’UTR lengthening.

By analyzing APA isoforms containing TEs at different portions of 3’UTR, we corroborated the recent finding by Gou et al. [45] that 3’UTR TEs are targeted for degradation in spermatogenesis. Using gene expression data from Miwi−/− mice [44], we further found that the degradation is through the piRNA-Miwi pathway. Thus, 3’UTR shortening can help genes evade the TE/piRNA/Miwi-based mRNA elimination during spermatogenesis. Previous studies have shown that TEs in 3’UTRs can play regulatory roles for mRNA metabolism [62–64] and some TEs can confer functional pAs to the host gene [65]. APA regulation in spermatogenesis can thus effectively permit evolution of TEs in 3’UTRs without inhibiting the expression of host genes, contributing to exaptation of TEs into 3’UTRs. Further studies are needed to elucidate how important this mechanism is for 3’UTR evolution. Also to be examined is whether some TE-containing aUTR sequences can give rise to piRNAs, which in turn regulate the host gene post-transcriptionally.

We found that 3’UTR shortening is coupled with upregulation of gene transcription and open state of chromatin, as indicated by RNAPII and H3K4me3 levels, respectively. Open chromatin has been suggested to cause widespread transcription in testis [30], leading to high complexity of the transcriptome. While this result is consistent with our previous finding implicating a role of transcriptional activity in APA regulation [53], the mechanism behind the coupling is unclear. One possibility is that permissive chromatin structure makes it more efficient to assemble the cleavage/polyadenylation machinery, leading to more usage of proximal pAs. However, other mechanisms involving specific factors to facilitate recruitment of the C/P machinery, such as that mediated by transcription factors [66], cannot be ruled out.

Widespread regulation of APA events in introns and internal exons suggests modulation of splicing activity during spermatogenesis, which is consistent with previous reports [31, 34–37]. Notably, the CDS-APA regulation between 3 and 4 weeks is reminiscent of the APA regulation by U1 snRNP inhibition [26, 27], where activation of pAs is largely biased to 5’ introns. Whether there is a localized shortage of U1 snRNP for certain genes leading to activation of intronic pAs needs to be examined in the future. Interestingly, genes with suppressed CDS-APA isoforms tend to be upregulated in expression (Fig. 7e) and have higher H3K4me3 levels (Fig. 7f), suggesting that open chromatin state may lead to efficient splicing, resulting in inhibition of intronic C/P.

We found global activation of uaRNAs during spermatogenesis (Fig. 8). These non-coding transcripts are generated by divergent promoters and are typically under the surveillance of the exosome [26, 29]. Their expression can significantly enrich the transcriptome during spermatogenesis, potentially impacting evolution of new genes [67]. uaRNA expression appears to be associated with open chromatin around the TSS and correlates with the expression of sense transcripts. It is not clear, however, why they are not eliminated by the nuclear exosome. Whether the function of exosome is suppressed in spermatogenesis or is overwhelmed by substantial activation of uaRNA expression needs to be addressed in the future.


Conclusions

This work represents the first comprehensive study of regulatory networks uncovered for a novel class of lncRNAs—vlincRNAs. The networks were initially obtained using the co-expression analysis based on SMS transcriptome profiling and then validated using two different approaches. This cross-validation approach confirmed the authenticity of these lncRNA regulatory networks and ensured validity of the following major conclusions derived from the analysis of these networks. First, a typical vlincRNA appears to function by regulating expression of multiple genes in cis and trans. Second, a vlincRNA can have both positive and negative effects on expression of different target genes. Third, vlincRNAs tend to regulate genes encoding certain functions related most notably to RNA processing, cell cycle, development, and adhesion. Fourth, the regulation depends on a mechanism based on co-localization, yet not necessarily direct interaction, of the vlincRNAs and their target genes in nucleus. In this regard, two other well-characterized lncRNAs NEAT1 and MALAT1 show similar mode of regulation. It is tempting to speculate therefore that regulation of multiple genes and doing so via proximity in the nucleus might be a common mode of lncRNA functionality. Furthermore, targeted knockdown of 2 vlincRNAs in stable cell lines has revealed biological importance of these transcripts and their regulatory networks in survival in response to genotoxic stress at least at the level of cultured cells similar to the results obtained previously by our group in a high-throughput screen [22]. However, additional studies are required to address of functionality of vlincRNAs, and lncRNAs in general, at the organismal level [12].


CONCLUSION

Human somatic stem cells, somatic tumor cells, and some adult cells may indeed express OCT4A mRNA at a basal level, compared with pluripotent cells. Nevertheless, the functional protein of OCT4A has not been reliably detected in the nonpluripotent cells. It is not clear whether the basal-level expression of OCT4A still be endowed other biological functions in nonpluripotent cells. However, the high-level expression of OCT4A protein still remains a property for pluripotent cells.

OCT4B is expressed at low levels in human somatic stem cells, tumor cells, adult tissues, as well as pluripotent cells. It is possible that OCT4B has diverse functions in different cells by various protein products. OCT4B is likely to play a role under stress response [ 20 ], and more detailed biological characterization of OCT4B should be explored in the future. It is interesting to note that OCT4B1 may be related to stemness [ 11 , 21 ], and investigations for OCT4B1 would contribute to stem cell research.

Each isoform of OCT4 reveals distinct sequence, thus OCT4A, OCT4B, and OCT4B1 may contain different RNA regulatory elements. Possibly, OCT4 isoforms may use different promoter and enhancer elements for their transcription. Therefore, OCT4 isoforms could be regulated respectively under the same conditions. However, it is possible that protein isoforms of OCT4 may have close connections with each other in biological functions due to the common sequence they shared. It is important to note that OCT4 isoforms are simultaneously expressed in some cells (e.g., human ES cells), hence it is conceivable that OCT4 isoforms may interact each other by competitive or synergistic interaction at transcriptional and/or post-transcriptional levels. The possibility and mechanisms remain unexplored.

The results summarized in this review suggest that OCT4 isoforms, not only OCT4A but also OCT4B and OCT4B1, contribute to the expression patterns and functions of OCT4 gene in a variety of human cells (Fig. 5). Like other crucial genes (such as FGF-2, VEGF, C-myc), OCT4 can generate transcript variants by alternative splicing and protein diversity by alternative translation initiation to overcome the limited number of genes in the genome and to perform multiple biological functions. To better understand OCT4, it is crucial to identify and distinguish its isoforms of OCT4 in expression patterns as well as function varieties in stem cell biology.

The schematic illustration of the relation of OCT4 isoforms to variant cells. Isoforms of OCT4 show the diversity in different cells. Abbreviations: iPS, induced pluripotent stem (cells) ES, embryonic stem (cells) PG, primordial germ (cells) GCTs, germ cell tumors.


Detecting Alternatively Spliced Transcript Isoforms from Single-Molecule Long-Read Sequences without a Reference Genome

Alternative splicing (AS) is a major source of transcript and proteome diversity, but examining AS in species without well-annotated reference genomes remains difficult. Research on both human and mouse has demonstrated the advantages of using Iso-Seq™ data for isoform-level transcriptome analysis, including the study of AS and gene fusion.

Researchers at the University of Florida applied Iso-Seq™ to investigate AS in Amborella trichopoda, a phylogenetically pivotal species that is sister to all other living angiosperms. Their data show that, compared with RNA-Seq data, the Iso-Seq™ platform provides better recovery on large transcripts, new gene locus identification, and gene model correction. Reference-based AS detection with Iso-Seq™ data identifies AS within a higher fraction of multi-exonic genes than observed for published RNA-Seq analysis (45.8% vs. 37.5%). These data demonstrate that the Iso-Seq™ approach is useful for detecting AS events. Using the Iso-Seq-defined transcript collection in Amborella as a reference, the researchers further describe a pipeline for detection of AS isoforms from PacBio Iso-Seq™ without using a reference sequence (de novo). Results using this pipeline show a 66-76% overall success rate in identifying AS events. This de novo AS detection pipeline provides a method to accurately characterize and identify bona fide alternatively spliced transcripts in any non-model system that lacks a reference genome sequence. Hence, this pipeline has huge potential applications and benefits to the broader biology community.


Abstract

While most long noncoding RNAs (lncRNAs) appear indistinguishable from mRNAs, having 5′ cap structures and 3′ poly(A) tails, recent work has revealed new formats. Rather than taking advantage of the canonical cleavage and polyadenylation for their 3′ end maturation, such lncRNAs are processed and stablized by a number of other mechanisms, including the RNase P cleavage to generate a mature 3′ end, or capped by snoRNP complexes at both ends, or by forming circular structures. Importantly, such lncRNAs have also been implicated in gene expression regulation in mammalian cells. Here, we highlight recent progress in our understanding of the biogenesis and function of lncRNAs without a poly(A) tail.

This paper is part of a directed issue entitled: The Non-coding RNA Revolution.


Novel TREM2 splicing isoform that lacks the V-set immunoglobulin domain is abundant in the human brain

Olena Korvatska, Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, WA, 98195, USA.

Department of Immunology, University of Washington, Seattle, Washington, USA

Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, Washington, USA

Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, Washington, USA

Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, Washington, USA

Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, Washington, USA

Department of Medicine, Division of Medical Genetics, University of Washington, Seattle, Washington, USA

Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, Washington, USA

Mental Illness Research, Education and Clinical Center (MIRECC), VA Puget Sound Medical Center, Seattle, Washington, USA

Geriatric Research, Education and Clinical Center (GRECC), VA Puget Sound Medical Center, Seattle, Washington, USA

Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, Washington, USA

Olena Korvatska, Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, WA, 98195, USA.

Funding information: National Institute of Health grants (P30AG013280 to O.K., 2R01 NS069719 to W.H.R.) Merit Review Award Number 101 CX001702.

Abstract

Triggering receptor expressed on myeloid cells 2 (TREM2) is an immunoglobulin-like receptor expressed by certain myeloid cells, such as macrophages, dendritic cells, osteoclasts, and microglia. In the brain, TREM2 plays an important role in the immune function of microglia, and its dysfunction is linked to various neurodegenerative conditions in humans. Ablation of TREM2 or its adaptor protein TYROBP causes polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy (also known as Nasu-Hakola disorder) with early onset of dementia, whereas some missense variants in TREM2 are associated with an increased risk of late-onset Alzheimer's disease. The human TREM2 gene is subject to alternative splicing, and its major, full-length canonic transcript encompasses 5 exons. Herein, we report a novel alternatively spliced TREM2 isoform without exon 2 (Δe2), which constitutes a sizable fraction of TREM2 transcripts and has highly variable inter-individual expression in the human brain (average frequency 10% range 3.7–35%). The protein encoded by Δe2 lacks a V-set immunoglobulin domain from its extracellular part but retains its transmembrane and cytoplasmic domains. We demonstrated Δe2 protein expression in TREM2-positive THP-1 cells, in which the expression of full-length transcript was precluded by CRISPR/Cas9 disruption of the exon 2 coding frame. Similar to the full-length TREM2, Δe2 is sorted to the plasma membrane and is subject to receptor shedding. In “add-back” experiments, Δe2 TREM2 had diminished capacity to restore phagocytosis of amyloid beta peptide and promote IFN-I response as compared to full-length TREM2. Our findings suggest that changes in the balance of two mutually exclusive TREM2 isoforms may modify the dosage of full-length transcript potentially weakening some TREM2 receptor functions in the human brain.

Table S1. Clinical and neuropathological characteristics of study subjects.

Supplementary Fig.S1. Nucleotide and protein sequence of canonical full length and De2 TREM2 isoforms.

Supplementary Fig.S2. Flow cytometry analysis settings for phagocytosis of pHrodo-labeled amyloid beta (Ab) peptide by THP-1 cells.

Supplementary Fig.S3. Expression of IFN I signature genes IFIH1 and IRF7 after stimulation of TREM2 KO THP-1 macrophages with poly(I:C) + IFNb.

Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.


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