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Model of cDNA synthesis that occurs in the absence (A) and presence (B and C) of RNA using standard TS oligo (A and B) vs. iso 3 TS oligo (C).
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The template switching PCR (TS-PCR) method of cDNA synthesis represents one of the most straightforward approaches to generating full length cDNA for sequencing efforts. However, when applied to very small RNA samples, such as those obtained from tens or hundreds of cells, this approach leads to high background and low cDNA yield due to concatameri...
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... key feature of our modification that enables this improvement of the TS-PCR method is the inclusion of isomeric nucleotide bases at the 5′ end of the TS oligo to inhibit MMLV-RT template switching activity after incorporation of the first TS oligo and thereby reduce or prevent TS oligo concatamerization (see Figure 1). We applied the modified method to construct cDNA libraries from glandular trichome secretory cells of tomato (Solanum) species and petunia, as well as from rhizome tips and elongation zones of scouring rush (Equisetum hyemale) and red rice (Oryza longistami- nata). ...
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... on results presented below, we developed the fol- lowing model that outlines how cDNA synthesis occurs in the absence and presence of RNA using standard TS oligo vs. iso 3 TS oligo (Figure 1). When a total RNA sample including mRNA transcripts is combined with oligo-dT primer sequence to prime first strand synthesis in the presence of TS oligo, the following can occur. ...
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... 1 -Oligo-dT primer anneals to the polyA tail of an mRNA transcript ( Figure 1B) and reverse transcriptase (RT) synthesizes first strand cDNA, or to some other polynucleotide entity in solution ( Figure 1A), such as a standard TS oligo. Upon reaching the 5′ end of the transcript (or other nucleotide entity), the terminal transferase activity of MMLV RT adds several additional bases, preferentially dC, to the 3′ end of the cDNA strand. ...
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... 1 -Oligo-dT primer anneals to the polyA tail of an mRNA transcript ( Figure 1B) and reverse transcriptase (RT) synthesizes first strand cDNA, or to some other polynucleotide entity in solution ( Figure 1A), such as a standard TS oligo. Upon reaching the 5′ end of the transcript (or other nucleotide entity), the terminal transferase activity of MMLV RT adds several additional bases, preferentially dC, to the 3′ end of the cDNA strand. ...
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... 2 -The rG 3 bases at the 3′ end of a standard TS oligo anneal to the polyC bases left by the RT at the 3′ end of the cDNA strand and RT switches templates, transcribing through the TS oligo DNA template ( Figure 1A and 1B). The TS oligo sequence and the extended 5′ portion of the oligo-dT primer are used as primer sites for cDNA amplification in the LD PCR step which fol- lows the RT reaction. ...
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... 3 -The terminal transferase activity of RT can add polyC bases to the end of a cDNA strand when using the standard TS oligo DNA as a template ( Figure 1A and 1B). 4 -Another TS oligo can then anneal and the process of TS oligo transcrip- tion and addition can continue indefinitely, adding a series of direct repeats of TS oligo sequence to the growing cDNA strand. Alterative Step 3 (iso 3 TS oligo is used) -Upon reaching the poly iso-nucleotide bases at the 5′ end of the iso 3 TS oligo, RT is inhibited from incorporating the corresponding nucleotides and reverse transcription terminates without addition of a polyC tail ( Figure 1C). ...
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... 3 -The terminal transferase activity of RT can add polyC bases to the end of a cDNA strand when using the standard TS oligo DNA as a template ( Figure 1A and 1B). 4 -Another TS oligo can then anneal and the process of TS oligo transcrip- tion and addition can continue indefinitely, adding a series of direct repeats of TS oligo sequence to the growing cDNA strand. Alterative Step 3 (iso 3 TS oligo is used) -Upon reaching the poly iso-nucleotide bases at the 5′ end of the iso 3 TS oligo, RT is inhibited from incorporating the corresponding nucleotides and reverse transcription terminates without addition of a polyC tail ( Figure 1C). Alterative Step 4 -No additional iso 3 TS oligo can anneal and concatamerization of TS oligo does not occur ( Figure 1C). ...
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... Step 3 (iso 3 TS oligo is used) -Upon reaching the poly iso-nucleotide bases at the 5′ end of the iso 3 TS oligo, RT is inhibited from incorporating the corresponding nucleotides and reverse transcription terminates without addition of a polyC tail ( Figure 1C). Alterative Step 4 -No additional iso 3 TS oligo can anneal and concatamerization of TS oligo does not occur ( Figure 1C). We developed this model as the result of performing experiments where we used TS-PCR to construct cell- type specific cDNA libraries from small numbers of iso- lated glandular trichome secretory cells from tomato and related Solanum species. ...
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... species contain sev- eral different types of glandular trichomes, some of which can only be isolated from other types by hand- selecting them from the surfaces of fresh and intact plant leaves using microcapillary probes under the gui- dance of a stereoscope, a time consuming and tedious procedure that yields only small numbers of isolated tri- chomes (100 and 500 gland samples were evaluated, see Figure 2). While constructing libraries from these very small samples, we encountered a significant flaw in the TS-PCR method as originally described [12]: a substan- tial quantity of cDNA spanning a size range that might be expected to represent a typical biological sample is synthesized in the absence of any starting RNA ( Figure 2A, lane 7; 2B, lane 1; 2C, lane 1; see Figure 1A). Differ- ences in level of cDNA synthesis for different quantities of starting material are difficult to distinguish after TS- PCR ( Figure 2A, lanes 1, 4 and 7). ...
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... constructing libraries from these very small samples, we encountered a significant flaw in the TS-PCR method as originally described [12]: a substan- tial quantity of cDNA spanning a size range that might be expected to represent a typical biological sample is synthesized in the absence of any starting RNA ( Figure 2A, lane 7; 2B, lane 1; 2C, lane 1; see Figure 1A). Differ- ences in level of cDNA synthesis for different quantities of starting material are difficult to distinguish after TS- PCR ( Figure 2A, lanes 1, 4 and 7). Substantial cDNA is generated in TS-PCR step even for negative control (Figure 2A, lane 7). ...
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... control cDNA synthesis reactions performed without RNA show high levels of cDNA synthesis for TS oligo as large as 2000 bp ( Figure 2B, lane 1), while the negative control cDNA synthesis performed using iso 3 TS oligo demonstrates almost complete suppression of artefactual synthesis of cDNA above 200 bp ( Figure 2B strong dependence on the presence of sample RNA to generate cDNA. ...
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... concatamerization of TS oligo occurs in cDNA syntheses from biological samples con- taining very small amounts of RNA ( Figure 2A, lanes 4-6), but appears to be reduced as the size of the biolo- gical sample increases ( Figure 2A, lanes 1-3). In samples containing either no RNA or very low numbers of mRNAs to serve as template for reverse transcription, RT activity is spuriously primed via primer-primer annealing ( Figure 1A). Although the efficiency of MMLV-RT terminal transferase activity toward uncapped RNA templates under the conditions used has been shown to be much lower than for capped tran- scripts (activity with DNA templates was not examined) [17], the low abundance of transcript templates increases the likelihood of terminal transferase addition of dC residues to the growing cDNA strand at the ends of any available template, including non-capped tem- plates. ...
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... the efficiency of MMLV-RT terminal transferase activity toward uncapped RNA templates under the conditions used has been shown to be much lower than for capped tran- scripts (activity with DNA templates was not examined) [17], the low abundance of transcript templates increases the likelihood of terminal transferase addition of dC residues to the growing cDNA strand at the ends of any available template, including non-capped tem- plates. After MMLV-RT has switched templates to the DNA TS oligo, the process is repeated and TS oligo is added indeterminately to the 3′ end of the growing cDNA strand (see Figures 1A/B and 3). ...
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... overall yield of high quality cDNA representing biological tran- scripts is also lower when the reverse transcriptase spends a significant proportion of its time synthesizing concatamers as opposed to unique cDNAs. We have developed a unique method to eliminate this concatamer-generated background problem that takes advantage of the expanded genetic alphabet [18,19] to prevent MMLV-RT from reaching the 5′ end of the TS oligo template after the initial template switch, thus pre- venting terminal transferase-mediated addition of dC bases at the end of the sequence complementary to the TS oligo template and subsequent concatamerization of TS oligo sequences ( Figure 1C). Stereoisomers of deoxy- cytosine and deoxyguanosine, referred to as isocytosine (iso-C) and isoguanosine,(iso-G), have the properties of natural nucleotide bases but have altered hydrogen bonding abilities which cause them to form nonstandard base pairs specifically with each other and discriminate against their naturally occurring analogs [17,19]. ...
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... 11 intervening bases are of unknown origin. See Figure 1A for a diagrammatic representation of the process whereby this type of concatamer may arise. Sequence example B shows 9 repeats of TS oligo concatamerized to a 178 base sequence with homology to nucleotide sequences from Vitus vinifera and Arabidopsis thaliana potentially representing a plant metalloendopeptidase. ...
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... oligo-dT reverse primer is also shown. See Figure 1B for a representation of the mechanism by which this type of concatameric sequence would be generated. occurred when using the unmodified TS oligo either with a small quantity of RNA template or without RNA template. ...
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... first strand cDNA synthesis using the iso 3 TS oligo while providing only the four naturally occurring nucleotide bases apparently causes MMLV-RT to prematurely stall before reaching the 5′ end of the iso 3 TS oligo template (see Figure 1). The great reduction of concatamer formation in these reactions suggested that misincorporation of natural bases by MMLV-RT opposite the synthetic bases was inhibited and that MMLV-RT terminal transferase activity was reduced, although we did not measure actual terminal transferase activity. ...
Citations
... 27 We incorporated in the protocol many features that had been proved to improve the quality and accuracy of RNA sequencing. In the cDNA reverse transcription step, we added 5 0 biotin blockers to the template-switching oligos (TSO) 34 and ran reactions with cycles at a higher temperature (50 C). 29 The former was to reduce the background concatemer formation (''hedgehog effects''), and the latter was to promote the unfolding of RNA secondary structure for a better yield of cDNA products. Moreover, the TSO contained a unique molecular identifier (UMI) that can be used at the data analysis step to correct PCR bias. ...
Generating large-scale, high-fidelity sequencing data is challenging and, furthermore, not much has been done to characterize adjuvants’ effects at the repertoire level. Thus, we introduced an IgSeq pipeline that standardized library prep protocols and data analysis functions for accurate repertoire profiling. We then studied systemically effects of CpG and Alum on the Ig heavy chain repertoire using the ovalbumin (OVA) murine model. Ig repertoires of different tissues (spleen and bone marrow) and isotypes (IgG and IgM) were examined and compared in IGHV mutation, gene usage, CDR3 length, clonal diversity, and clonal selection. We found Ig repertoires of different compartments exhibited distinguishable profiles at the non-immunized steady state, and distinctions became more pronounced upon adjuvanted immunizations. Notably, Alum and CpG effects exhibited different tissue- and isotype-preferences. The former led to increased diversity of abundant clones in bone marrow, and the latter promoted the selection of IgG clones in both tissues.
... The oligo-dT (Smart-dTV30) primer, template switching oligo (TSO) and preamplification (IS-PCR) primers were all modified with a 5'-end biotin molecule (Table 1. This is crucial to increase cDNA yield by avoiding concatenation of TSOs after the first stand reaction (Kapteyn et al. 2010, Turchinovich et al. 2014). The lysis buffer contained a final concentration of 0.2 % Triton -X100 (Sigma), 1 U/μL RNAse inhibitor (cat. ...
Single-cell transcriptomics is a vital tool for unraveling metabolism and tissue diversity in model organisms. Its potential for elucidating the ecological roles of microeukaryotes, especially non-model ones, remains largely unexplored. This study employed the Smart-seq2 protocol on Ochromonas triangulata, a microeukaryote lacking a reference genome, showcasing how transcriptional states align with growth phases. Unexpectedly, a third transcriptional state was identified, across both growth phases. Metabolic mapping revealed a down-regulation trend in path-ways associated with ribosome functioning, CO2 fixation, and carbohydrate catabolism from fast to slow growth to the third transcriptional state. Using carry-over rRNA reads, taxonomic identity of Ochromonas triangulata was re-confirmed and distinct bacterial communities associated with transcriptional states were identified. This study underscores single-cell transcriptomics as a powerful tool for characterizing metabolic states in microeukaryotes without a reference genome, offering in-sights into unknown physiological states and individual-level interactions with different bacterial taxa. This approach holds broad applicability for uncovering ecological roles, surpassing alternative methods like metagenomics or metatranscriptomics.
... It appends a few Cs to the 3′ end of the newly synthesized DNA at the end of the RNA template. In the presence of G-tailed single-strand DNA (ssDNA), MMLV-RT can switch the template and polymerize the complementary strand of incoming ssDNA (10,11). This template-switching reaction has been widely used in various studies to analyze RNA-seq (12). ...
Development and characterization of a new enzyme reaction benefit modern biotechnologies. Here, we report a novel CIS (clamping-mediated incorporation of single-stranded DNA with concomitant DNA synthesis) reaction catalyzed by Taq polymerase. In the reaction, a single-stranded DNA (ssDNA) with 3′ Cs is attached with a preformed 3′ G-tail of double-stranded DNA (dsDNA); DNA syntheses starting from both 3′ ends result in the incorporation of ssDNA. A 3′ G-tail length of 3 nucleotides adequately supports this reaction, indicating that Taq polymerase can clump short Watson-Crick base pairs as short as three pairs and use them to initiate DNA polymerization. The reverse transcriptase from Molony murine leukemia virus catalyzes strand displacement synthesis and produces flapped-end DNA, whereas the reaction by Taq polymerase involves the nick translation. These new reaction properties may be beneficial for the development of new molecular tools applicable in various fields. Apart from its CIS reaction activity, we also report that Taq polymerase has the undesirable characteristic of removing 5' fluorescent labels from dsDNA. This characteristic may have compromised various experiments involving the preparation of fluorescently-labeled dsDNA by PCR for a long time.
... The module then groups these annotations into recurring patterns of how these building blocks are arranged and provides an overview of the relative contributions of each class of such arrangements to the library. This allows the user to monitor cDNA integrity, for example, from 10X Chromium beads (Supplementary Fig. S2B, C), and enables to detect and subsequently mitigate potential primer and Template Switch Oligo (TSO) concatenations as described in [29]. ...
Background
Spatial sequencing methods increasingly gain popularity within RNA biology studies. State-of-the-art techniques quantify messenger RNA expression levels from tissue sections and at the same time register information about the original locations of the molecules in the tissue. The resulting data sets are processed and analyzed by accompanying software that, however, is incompatible across inputs from different technologies.
Findings
Here, we present spacemake, a modular, robust, and scalable spatial transcriptomics pipeline built in Snakemake and Python. Spacemake is designed to handle all major spatial transcriptomics data sets and can be readily configured for other technologies. It can process and analyze several samples in parallel, even if they stem from different experimental methods. Spacemake's unified framework enables reproducible data processing from raw sequencing data to automatically generated downstream analysis reports. Spacemake is built with a modular design and offers additional functionality such as sample merging, saturation analysis, and analysis of long reads as separate modules. Moreover, spacemake employs novoSpaRc to integrate spatial and single-cell transcriptomics data, resulting in increased gene counts for the spatial data set. Spacemake is open source and extendable, and it can be seamlessly integrated with existing computational workflows.
... The rGrG+G is included at the 3ʹ end to increase the affinity of the TSO toward the nontemplated CCC residues added to the ends of RT products, which will increase the success rate of template switching 47 . The iCiGiC at the 5ʹ end of the TSO can inhibit the reverse transcriptase from extending the cDNA beyond the TSO, which avoids the formation of concatamers of TSOs and increases cDNA yield 48 . Moreover, the oligo is 3′-aminated (/NH 2 -C3-3′) to block it from working as an RT primer in RT. ...
Poly(A) tails are added to the 3′ ends of most mRNAs in a non-templated manner and play essential roles in post-transcriptional regulation, including mRNA export, stability and translation. Measuring poly(A) tails is critical for understanding their regulatory roles in almost every aspect of biological and medical studies. Previous methods for analyzing poly(A) tails require large amounts of input RNA (microgram-level total RNA), which limits their application. We recently developed a poly(A) inclusive full-length RNA isoform-sequencing method (PAIso-seq) at single-oocyte-level sensitivity (a single mammalian oocyte contains ~0.5 ng of total RNA) based on PacBio sequencing that enabled accurate measurement of the poly(A) tail length and non-A residues within the body of poly(A) tails along with the full-length cDNA, providing the opportunity to study precious in vivo samples with very limited input material. Here, we describe a detailed protocol for PAIso-seq library preparation from single mouse oocytes or bulk oocyte samples. In addition, we provide a complete bioinformatic pipeline to perform the analysis from the raw data to downstream analysis. The minimum time required is ~14.5 h for PAIso-seq double-stranded cDNA preparation, 2 d for PacBio sequencing in HiFi mode and 8 h for the initial data analysis. Poly(A) inclusive full-length RNA isoform sequencing is a method for measuring poly(A) tail length and composition at single-mammalian-oocyte (subnanogram)-level sensitivity.
... Table 4), nuclease-free water till 35 µl and subsequent incubation at 42°C for 2 hours. The TSO was designed with two isodeoxynucleotides at the 5' end to prevent TSO concatemerization and three riboguanosines at the 3' end for increased binding affinity to the appended deoxycytidines (property of the Takara reverse transcriptase) (80,81). The single-stranded cDNA was immediately purified using Zymo's RNA Clean & Concentrator kit (#R1016) using Zymo's appended protocol to purify fragments >200 nucleotides and was eluted in 10 µl elution buffer. ...
Memory B cells (MBCs) and plasma antibodies against Plasmodium falciparum (Pf) merozoite antigens are important components of the protective immune response against malaria. To gain understanding of how responses against Pf develop in these two arms of the humoral immune system, we evaluated MBC and antibody responses against the most abundant merozoite antigen, full-length Pf merozoite surface protein 1 (PfMSP1FL), in individuals from a region in Uganda with high Pf transmission. Our results showed that PfMSP1FL-specific B cells in adults with immunological protection against malaria were predominantly IgG⁺ classical MBCs, while children with incomplete protection mainly harbored IgM⁺ PfMSP1FL-specific classical MBCs. In contrast, anti-PfMSP1FL plasma IgM reactivity was minimal in both children and adults. Instead, both groups showed high plasma IgG reactivity against PfMSP1FL, with broadening of the response against non-3D7 strains in adults. The B cell receptors encoded by PfMSP1FL-specific IgG⁺ MBCs carried high levels of amino acid substitutions and recognized relatively conserved epitopes on the highly variable PfMSP1 protein. Proteomics analysis of PfMSP119-specific IgG in plasma of an adult revealed a limited repertoire of anti-MSP1 antibodies, most of which were IgG1 or IgG3. Similar to B cell receptors of PfMSP1FL-specific MBCs, anti-PfMSP119 IgGs had high levels of amino acid substitutions and their sequences were predominantly found in classical MBCs, not atypical MBCs. Collectively, these results showed evolution of the PfMSP1-specific humoral immune response with cumulative Pf exposure, with a shift from IgM⁺ to IgG⁺ B cell memory, diversification of B cells from germline, and stronger recognition of PfMSP1 variants by the plasma IgG repertoire.
... The first FLAM-Seq protocol contained a large number of concatemer sequences related to the template switch oligo (TSO) used for reverse transcription, which could be efficiently alleviated by chemically modifying the 5'-end using non-natural nucleotides 360 and could be related to coverage at the 5'-ends. Although this type of analysis was in some cases possible using previous methods 157 , FLAM-Seq is the preferred method. ...
Der Ribonukleinsäure (RNS) Stoffwechsel umfasst verschiedene Schritte, beginnend mit der Transkription der RNS über die Translation bis zum RNA Abbau. Poly(A) Schwänze befinden sich am Ende der meisten der Boten-RNS, schützen die RNA vor Abbau und stimulieren Translation. Die Deadenylierung von Poly(A) Schwänzen limitiert den Abbau von RNS. Bisher wurde RNS Abbau meist im Kontext von cytoplasmatischen Prozessen untersucht, ob und wie RNS Deadenylierung und Abbau in Nukleus erfolgen ist bisher unklar. Es wurde daher eine neue Methode zur genomweiten Bestimmung von Poly(A) Schwanzlänge entwickelt, welche FLAM-Seq genannt wurde. FLAM-Seq wurde verwendet um Zelllinien, Organoide und C. elegans RNS zu analysieren und es wurde eine signifikante Korrelation zwischen 3’-UTR und Poly(A) Länge gefunden, sowie für viele Gene ein Zusammenhang von alternativen 3‘-UTR Isoformen und Poly(A) Länge. Die Untersuchung von Poly(A) Schwänzen von nicht-gespleißten RNS Molekülen zeige, dass deren Poly(A) Schwänze eine Länge von mehr als 200 nt hatten. Die Analyse wurde durch eine Inhibition des Spleiß-Prozesses validiert. Die Verwendung von Methoden zur Markierung von RNS, welche die zeitliche Auflösung der RNS Prozessierung ermöglicht, deutete auf eine Deadenylierung der Poly(A) Schwänze schon wenige Minuten nach deren Synthesis hin. Die Analyse von subzellulären Fraktionen zeigte, dass diese initiale Deadenylierung ein Prozess im Nukleus ist. Dieser Prozess ist gen-spezifisch und Poly(A) Schwänze von bestimmten Typen von Transkripten, wie nuklearen langen nicht-kodierende RNS Molekülen waren nicht deadenyliert. Um Enzyme zu identifizieren, welche die Deadenylierung im Zellkern katalysieren, wurden verschiedene Methoden wie RNS-abbauende Cas Systeme, siRNAs oder shRNA Zelllinien verwendet. Trotz einer effizienten Reduktion der RNS Expression entsprechender Enzymkomplexe konnten keine molekularen Phänotypen identifiziert werden welche die Poly(A) Länge im Zellkern beeinflussen.
... TSRT can also result in TSO chaining, wherein once RT reaches the 5′ end of the TSO, it performs another round of non-templated nucleotide addition, allowing another TSO to bind. The prevalence of the resulting TSO concatemers can be reduced by 3′ modification of the TSO with non-natural nucleotides (Kapteyn et al., 2010) or biotin (Turchinovich et al., 2014). ...
Transcription start site (TSS) selection influences transcript stability and translation as well as protein sequence. Alternative TSS usage is pervasive in organismal development, is a major contributor to transcript isoform diversity in humans, and is frequently observed in human diseases including cancer. In this review, we discuss the breadth of techniques that have been used to globally profile TSSs and the resulting insights into gene regulation, as well as future prospects in this area of inquiry.
... A common approach uses the terminal transferase activity of certain reverse transcriptase enzymes to facilitate a ''template-switch'' from the original mRNA to a second defined oligonucleotide (Picelli et al., 2013). Although simple to implement, this process has the potential to be highly inefficient, leading to the loss of molecules that have been converted to cDNA but not successfully tagged with a secondary PCR priming site (Islam et al., 2012;Kapteyn et al., 2010;Zajac et al., 2013). ...
High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S³ (“Second-Strand Synthesis”), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S³ increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S³ to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.
... TSS mapping methods incorporating TSRT include 5 ′ serial analysis of gene expression (5 ′ SAGE) (Zhang and Dietrich 2005), nano-cap analysis of gene expression (nanoCAGE 2010(nanoCAGE /2017 (Plessy et al. 2010;Poulain et al. 2017), single-cell tagged reverse transcription (STRT) (Islam et al. 2011), RNA annotation and mapping of promoters for the analysis of gene expression (RAMPAGE) (Batut et al. 2013), Tn5Prime (Cole et al. 2018), and parallel analysis of RNA 5 ′ ends from low input (nanoPARE) (Schon et al. 2018). Although TSRT provides additional specificity for mRNA 5 ′ ends, library complexity is often limited by artifacts such as concatemerization of TSOs from secondary template-switching events, erroneous template switching, and high PCR cycle requirements (Kapteyn et al. 2010;Turchinovich et al. 2014). Some TSRT-based methods (RAM-PAGE, nanoCAGE 2017, and nanoPARE) also require custom sequencing primers, complicating pooling of other sample types in the same sequencing lane. ...
Accurate mapping of transcription start sites (TSSs) is key for understanding transcriptional regulation. However, current protocols for genome-wide TSS profiling are laborious and/or expensive. We present Survey of TRanscription Initiation at Promoter Elements with high-throughput sequencing (STRIPE-seq), a simple, rapid, and cost-effective protocol for sequencing capped RNA 5′ ends from as little as 50 ng total RNA. Including depletion of uncapped RNA and reaction cleanups, a STRIPE-seq library can be constructed in about 5 h. We show application of STRIPE-seq to TSS profiling in yeast and human cells and show that it can also be effectively used for quantification of transcript levels and analysis of differential gene expression. In conjunction with our ready-to-use computational workflows, STRIPE-seq is a straightforward, efficient means by which to probe the landscape of transcriptional initiation.
