Dominant Isoform in Alternative Splicing in HeLa S3 Cell Line Revealed by Single-cell RNA-seq

2016 ◽  
Author(s):  
Zhikun Zhao ◽  
Jing Tu ◽  
Zuhong Lu ◽  
Shiping Liu
Keyword(s):  
Alternative Splicing ◽  
Single Cell ◽  
Cell Line ◽  
Rna Seq ◽  
Hela S3

scholarly journals SAT-298 Integrative Single-Cell Transcriptomic and Epigenomic Landscape of Mouse Anterior Pituitary Cell Types

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Frederique Murielle Ruf-Zamojski ◽  
Michel A Zamojski ◽  
German Nudelman ◽  
Yongchao Ge ◽  
Natalia Mendelev ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Line ◽  
Anterior Pituitary ◽  
Cell Types ◽  
Chromatin Accessibility ◽  
Pituitary Cell ◽  
Integrated Analysis ◽  
Pituitary Cells ◽  
Rna Seq ◽  
Cell Type

Abstract The pituitary gland is a critical regulator of the neuroendocrine system. To further our understanding of the classification, cellular heterogeneity, and regulatory landscape of pituitary cell types, we performed and computationally integrated single cell (SC)/single nucleus (SN) resolution experiments capturing RNA expression, chromatin accessibility, and DNA methylation state from mouse dissociated whole pituitaries. Both SC and SN transcriptome analysis and promoter accessibility identified the five classical hormone-producing cell types (somatotropes, gonadotropes (GT), lactotropes, thyrotropes, and corticotropes). GT cells distinctively expressed transcripts for Cga, Fshb, Lhb, Nr5a1, and Gnrhr in SC RNA-seq and SN RNA-seq. This was matched in SN ATAC-seq with GTs specifically showing open chromatin at the promoter regions for the same genes. Similarly, the other classically defined anterior pituitary cells displayed transcript expression and chromatin accessibility patterns characteristic of their own cell type. This integrated analysis identified additional cell-types, such as a stem cell cluster expressing transcripts for Sox2, Sox9, Mia, and Rbpms, and a broadly accessible chromatin state. In addition, we performed bulk ATAC-seq in the LβT2b gonadotrope-like cell line. While the FSHB promoter region was closed in the cell line, we identified a region upstream of Fshb that became accessible by the synergistic actions of GnRH and activin A, and that corresponded to a conserved region identified by a polycystic ovary syndrome (PCOS) single nucleotide polymorphism (SNP). Although this locus appears closed in deep sequencing bulk ATAC-seq of dissociated mouse pituitary cells, SN ATAC-seq of the same preparation showed that this site was specifically open in mouse GT, but closed in 14 other pituitary cell type clusters. This discrepancy highlighted the detection limit of a bulk ATAC-seq experiment in a subpopulation, as GT represented ~5% of this dissociated anterior pituitary sample. These results identified this locus as a candidate for explaining the dual dependence of Fshb expression on GnRH and activin/TGFβ signaling, and potential new evidence for upstream regulation of Fshb. The pituitary epigenetic landscape provides a resource for improved cell type identification and for the investigation of the regulatory mechanisms driving cell-to-cell heterogeneity. Additional authors not listed due to abstract submission restrictions: N. Seenarine, M. Amper, N. Jain (ISMMS).

Single-cell RNA-seq reveals lincRNA expression differences in Hela-S3 cells

2016 ◽  
Vol 39 (3) ◽  
pp. 359-366 ◽  
Author(s):  
Jie Wang ◽  
Bhaskar Roy
Keyword(s):  
Single Cell ◽  
Rna Seq ◽  
Hela S3 ◽  
Hela S3 Cells

scholarly journals Single Cell RNA-Seq Characterises Pre-Leukemic Transformation Driven By CEBPA N321D in the Hoxb8-FL Cell Line

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3887-3887
Author(s):  
Moosa Qureshi ◽  
Fernando Calero-Nieto ◽  
Iwo Kucinski ◽  
Sarah Kinston ◽  
George Giotopoulos ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Single Cell ◽  
Cell Line ◽  
Single Cells ◽  
Mutant Form ◽  
Rna Seq ◽  
Wild Type ◽  
Leukemic Transformation

Abstract The C/EBPα transcription factor plays a pivotal role in myeloid differentiation and E2F-mediated cell cycle regulation. Although CEBPA mutations are common in acute myeloid leukaemia (AML), little is known regarding pre-leukemic alterations caused by mutated CEBPA. Here, we investigated early events involved in pre-leukemic transformation driven by CEBPA N321D in the LMPP-like cell line Hoxb8-FL (Redecke et al., Nat Methods 2013), which can be maintained in vitro as a self-renewing LMPP population using Flt3L and estradiol, as well as differentiated both in vitro and in vivo into myeloid and lymphoid cell types. Hoxb8-FL cells were retrovirally transduced with Empty Vector (EV), wild-type CEBPA (CEBPA WT) or its N321D mutant form (CEBPA N321D). CEBPA WT-transduced cells showed increased expression of cd11b and SIRPα and downregulation of c-kit, suggesting that wild-type CEBPA was sufficient to promote differentiation even under LMPP growth conditions. Interestingly, we did not observe the same phenotype in CEBPA N321D-transduced cells. Upon withdrawal of estradiol, both EV and CEBPA WT-transduced cells differentiated rapidly into a conventional dendritic cell (cDC) phenotype by day 7 and died within 12 days. By contrast, CEBPA N321D-transduced cells continued to grow for in excess of 56 days, with an initial cDC phenotype but by day 30 demonstrating a plasmacytoid dendritic cell precursor phenotype. CEBPA N321D-transduced cells were morphologically distinct from EV-transduced cells. To test leukemogenic potential in vivo, we performed transplantation experiments in lethally irradiated mice. Serial monitoring of peripheral blood demonstrated that Hoxb8-FL derived cells had disappeared by 4 weeks, and did not reappear. However, at 6 months CEBPA N321D-transduced cells could still be detected in bone marrow in contrast to EV-transduced cells but without any leukemic phenotype. To identify early events involved in pre-leukemic transformation, the differentiation profiles of EV, CEBPA WT and CEBPA N321D-transduced cells were examined with single cell RNA-seq (scRNA-seq). 576 single cells were taken from 3 biological replicates at days 0 and 5 post-differentiation, and analysed using the Automated Single-Cell Analysis Pipeline (Gardeux et al., Bioinformatics 2017). Visualisation by t-SNE (Fig 1) demonstrated: (i) CEBPA WT-transduced cells formed a distinct cluster at day 0 before withdrawal of estradiol; (ii) CEBPA N321D-transduced cells separated from EV and CEBPA WT-transduced cells after 5 days of differentiation, (iii) two subpopulations could be identified within the CEBPA N321D-transduced cells at day 5, with a cluster of five CEBPA N321D-transduced single cells distributed amongst or very close to the day 0 non-differentiated cells. Differential expression analysis identified 224 genes upregulated and 633 genes downregulated specifically in the CEBPA N321D-transduced cells when compared to EV cells after 5 days of differentiation. This gene expression signature revealed that CEBPA N321D-transduced cells switched on a HSC/MEP/CMP transcriptional program and switched off a myeloid dendritic cell program. Finally, in order to further dissect the effect of the N321D mutation, the binding profile of endogenous and CEBPA N321D was compared by ChIP-seq before and after 5 days of differentiation. Integration with scRNA-seq data identified 160 genes specifically downregulated in CEBPA N321D-transduced cells which were associated with the binding of the mutant protein. This list of genes included genes previously implicated in dendritic cell differentiation (such as NOTCH2, JAK2), as well as a number of genes not previously implicated in the evolution of AML, representing potentially novel therapeutic targets. Disclosures No relevant conflicts of interest to declare.

scholarly journals DOP23 Single-cell RNA sequencing identifies an important role for class I histone-deacetylase enzymes in intestinal myofibroblasts from patients with Crohn’s Disease strictures

2021 ◽  
Vol 15 (Supplement_1) ◽  
pp. S062-S062
Author(s):  
A Lewis ◽  
B Pan-Castillo ◽  
G Berti ◽  
C Felice ◽  
H Gordon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cell Line ◽  
Rna Sequencing ◽  
Histone Deacetylase ◽  
Chromatin Accessibility ◽  
Collagen I ◽  
Class I ◽  
Rna Seq ◽  
Single Cell Rna Sequencing

Abstract Background Histone-deacetylase (HDAC) enzymes are a broad class of ubiquitously expressed enzymes that modulate histone acetylation, chromatin accessibility and gene expression. In models of Inflammatory bowel disease (IBD), HDAC inhibitors, such as Valproic acid (VPA) are proven anti-inflammatory agents and evidence suggests that they also inhibit fibrosis in non-intestinal organs. However, the role of HDAC enzymes in stricturing Crohn’s disease (CD) has not been characterised; this is key to understanding the molecular mechanism and developing novel therapies. Methods To evaluate HDAC expression in the intestine of SCD patients, we performed unbiased single-cell RNA sequencing (sc-RNA-seq) of over 10,000 cells isolated from full-thickness surgical resection specimens of non-SCD (NSCD; n=2) and SCD intestine (n=3). Approximately, 1000 fibroblasts were identified for further analysis, including a distinct cluster of myofibroblasts. Changes in gene expression were compared between myofibroblasts and other resident intestinal fibroblasts using the sc-RNA-seq analysis pipeline in Partek. Changes in HDAC expression and markers of HDAC activity (H3K27ac) were confirmed by immunohistochemistry in FFPE tissue from patient matched NSCD and SCD intestine (n=14 pairs). The function of HDACs in intestinal fibroblasts in the CCD-18co cell line and primary CD myofibroblast cultures (n=16 cultures) was assessed using VPA, a class I HDAC inhibitor. Cells were analysed using a variety of molecular techniques including ATAC-seq, gene expression arrays, qPCR, western blot and immunofluorescent protein analysis. Results Class I HDAC (HDAC1, p= 2.11E-11; HDAC2, p= 4.28E-11; HDAC3, p= 1.60E-07; and HDAC8, p= 2.67E-03) expression was increased in myofibroblasts compared to other intestinal fibroblasts subtypes. IHC also showed an increase in the percentage of stromal HDAC2 positive cells, coupled with a decrease in the percentage of H3K27ac positive cells, in the mucosa overlying SCD intestine relative to matched NSCD areas. In the CCD-18co cell line and primary myofibroblast cultures, VPA reduced chromatin accessibility at Collagen-I gene promoters and suppressed their transcription. VPA also inhibited TGFB-induced up-regulation of Collagen-I, in part by inhibiting TGFB1|1/SMAD4 signalling. TGFB1|1 was identified as a mesenchymal specific target of VPA and siRNA knockdown of TGFB1|1 was sufficient suppress TGFB-induced up-regulation of Collagen-I. Conclusion In SCD patients, class I HDAC expression is increased in myofibroblasts. Class I HDACs inhibitors impair TGFB-signalling and inhibit Collagen-I expression. Selective targeting of TGFB1|1 offers the opportunity to increase treatment specificity by selectively targeting meschenymal cells.

Investigation of the Mechanism of Idelalisib Resistance in the Follicular Lymphoma WSU-Fsccl Cell Line

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2482-2482 ◽  
Author(s):  
Rick Sorensen ◽  
Sarah Meadows ◽  
Anella Yahiaoui ◽  
Li Li ◽  
Peng Yue ◽  
...  
Keyword(s):  
Western Blot ◽  
Growth Inhibition ◽  
Follicular Lymphoma ◽  
Single Cell ◽  
Cell Line ◽  
Wnt Pathway ◽  
Parental Line ◽  
Rna Seq ◽  
Resistance Mutations ◽  
Cell Clones

Abstract Idelalisib (Zydelig®), a first-in-class, selective, oral inhibitor of PI3Kd, is approved in the US and EU for the treatment of patients with follicular lymphoma (FL) who have received at least 2 prior systemic therapies based on the outcome of a phase 2 clinical trial demonstrating an ORR of 54% (Gopal AK, et al. ASH 2014, Abstract 1708; Zydelig® SmPC, Mar 2015). Complete responses are rare (6%) and patients ultimately relapse leading to an urgent need to understand mechanisms of resistance (MOR) to idelalisib. We report here the MOR identified from 3 sets of the transformed FL cell line WSU-FSCCL which were made resistant to idelalisib by continuous in vitro exposure. Methods: Idelalisib resistance was established by continuous passaging of a clonal isolate of WSU-FSCCL in the presence of 1 μM idelalisib. Growth inhibition to idelalisib or other inhibitors was performed using CellTiter Glo viability assay (Promega) at 96 h. Clonal isolates of idelalisib sensitive (FSCCLS) and idelalisib resistant (FSCCLR) cell lines were generated through two rounds of single cell limiting dilution. Characterization of mutations and gene expression in FSCCLS and FSCCLR clones was done by whole exome sequencing (WES, GENEWIZ) and RNA-Seq (Expression Analysis), respectively. Whole cell lysates were analyzed by Simple Western using Peggy Sue (Protein Simple) or SDS-PAGE and Western blot. Results: WSU-FSCCL were highly sensitive to idelalisib and the pan-PI3K inhibitor GDC-0941 with an EC50 of 140 and 180 nM, respectively, indicating that cell viability is driven by PI3Kd, while profiling of BTK inhibitors showed continued lack of activity. FSCCLR showed a loss of sensitivity to idelalisib (1 μM) with a maximal growth inhibition of 16% vs. 85% for parental line (FSCCLS). WES analysisrevealed PIK3CA resistance mutations in 3 independently generated sets of FSCCLR single cell clones, while no PIK3CD, PIK3CB, or PIK3CG resistance mutations were observed. PIK3CA was mutated at N345K (11/11 clones), P539R (3/3 clones) and E970K (3/3 clones). PI3K isoform profiling showed no alterations in expression of any of the four p110 isoforms. Idelalisib activity on PIK3CA mutant clone viability was dramatically shifted (EC50 >5 μM). The sensitivity of PIK3CA mutant FSCCLR to the PI3Kα- specific inhibitor BYL719 was not increased compared to FSCCLS (EC50 > 1.5 μM).Interestingly the combination of idelalisib treatment with BYL719 (0.5 μM) restored the sensitivity of FSCCLR to a concentration in the range of parental cells (EC50 = 0.066 μM, Figure 1). A second set of FSCCLR clones (set 2) were PI3KCA WT and had a 2.7-fold upregulation of PTEN. A survey of compensatory pathway activation revealed upregulation of Src family kinase (SFK) phosphorylation (p-SFK Y416) and specifically of p-Hck Y411 and p-Lyn Y396. SFK phosphorylation was sensitive to treatment with the SFK inhibitor dasatinib. Set 2 FSCCLR were slightly less sensitive to dasatinib (EC50 = 0.058 μM) compared to FSCCLS (EC50 = 0.034 μM). Interestingly, addition of 30 nM dasatinib (EC25 in FSCCLS) to FSCCLR increased the sensitivity of set 2 FSCCLR to idelalisib (EC50 = 0.95 μM) (Figure 2). FSCCLR clones were more resistant to the Syk inhibitor entospletinib (ENTO) (EC50 > 10 μM) as compared to FSCCLS (EC50 = 0.18 μM). Profiling of idelalisib activity on set 2 FSCCLR clones showed resistance to idelalisib as expected (EC50 > 10, μM); the addition of entospletinib at a clinically relevant concentration (0.68 μM), resulted in greater sensitivity than use of either single agent alone (EC50 = 2.27 μM ) (Figure 3). RNA-Seq analysis of the FSCCLR PIK3CA WT single cell clones additionally revealed that a subset of clones (set 3) upregulated a set of WNT pathway genes. Western blot analysis of set 3 FSCCLR showed upregulation of downstream markers of the canonical WNT pathway, including LEF1/TCF, c-Jun, β-catenin, c-Myc and p-GSK3β. Conclusions: Mechanisms of idelalisib resistance in the follicular lymphoma WSU-FSCCL cell line were independent of alterations in p110d but included the likely gain of function mutations in the PIK3CA gene and activation of SFK and WNT pathways. The sensitivity to idelalisib in the resistant clones could be re-established by the combination of idelalisib with inhibitors of compensatory pathways: BYL719 in PIK3CA mutant clones and dasatinib or entospletinib in SFK activated clones. Disclosures Sorensen: Gilead Sciences: Employment, Other: Share holder. Meadows:Gilead Sciences: Employment, Other: Share holder. Yahiaoui:Gilead Sciences: Employment, Other: Share holder. Li:Gilead Sciences: Employment, Other: Share holder. Yue:Gilead Sciences: Employment, Other: Share holder. Kashishian:Gilead Sciences: Employment, Other: Share holder. Queva:Gilead Sciences: Other: Share holder. Tannheimer:Gilead Sciences: Employment, Other: Share holder.

Comprehensive Bulk and Single Cell Transcriptomic Characterization of SF3B1 Mutation Reveals Its Pleiotropic Effects in Chronic Lymphocytic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2906-2906 ◽  
Author(s):  
Jean Fan ◽  
Lili Wang ◽  
Angela N Brooks ◽  
Youzhong Wan ◽  
Donna S Neuberg ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Single Cell ◽  
Single Cells ◽  
Lymphocytic Leukemia ◽  
Rna Seq ◽  
Wild Type ◽  
Mutation Status ◽  
Total Rna ◽  
Gene Sets

Abstract Large-scale sequencing efforts have identified SF3B1 as arecurrently mutated gene in chronic lymphocytic leukemia (CLL). While SF3B1 mutations have been associated with adverse clinical outcome in CLL, mechanistic understanding of its role in the oncogenic phenotype remains lacking. We therefore undertook a comprehensive transcriptomic characterization of CLL in relation to SF3B1 mutation status at both bulk and single cell levels. We first profiled bulk mature poly-A selected RNA by sequencing (RNA-seq) from 37 CLLs (13 SF3B1 wild-type, 24 mutated). After identifying and classifying splice alterations using the tool JuncBASE, we found SF3B1 mutation to be associated with increased alternative splicing, with the most pervasive changes in 3' splice site selection. 304 alternatively spliced events were significantly associated with SF3B1 mutation, 4 of which we validated by qRT-PCR in 20 independent CLL samples with known SF3B1 mutation status. We further identified 1963 differentially expressed genes (q < 0.2) associated with SF3B1 mutation. By gene set enrichment analysis, SF3B1 mutation appeared to impact a variety of cancer and CLL-associated gene pathways, including DNA damage response, apoptosis regulation, chromatin remodeling, RNA processing, and Notch activation (q < 0.01). ~20% of these gene sets were also found to be significantly enriched for genes exhibiting alternative splicing in association with SF3B1 mutation. As SF3B1 acts at the level of pre-mRNA, we also performed bulk RNA-seq with total RNA libraries generated from 5 CLLs (2 SF3B1 wild-type, 3 with the common K700E mutation). We again observed an enrichment of 3' splice site changes, along with ~30% overlap of differentially expressed genes, and ~16% overlap of enriched gene sets with the aforementioned poly-A data analysis. One differentially over-expressed gene associated with SF3B1 mutation unique to this total RNA data analysis and validated by total RNA qPCR of independent CLL samples was TERC, an essential RNA component of telomerase that serves as a replication template during telomeric elongation. TERC is a non-polyadenylated transcript and thus was undetected by our previous poly-A selected RNA-seq and by targeted qRT-PCR of oligo dT-generated cDNA. Recent reports have highlighted the involvement of the spliceosome in telomerase RNA processing, and shorter telomere length of CLLs with SF3B1 mutation. Thus, although further investigation will be needed, our analyses suggest a potential mechanism by which SF3B1 mutation contributes to aberrant regulation of telomerase activity. Since SF3B1 is commonly found as a subclonal mutation in CLL, and because signals obtained from bulk analyses reflect only the average characteristics of the population, we assessed the transcriptomic effects of SF3B1 mutation in single cells within a subset of CLL cases. We developed a novel and sensitive microfluidic approach that performs multiplexed targeted amplification of RNA to simultaneously detect somatic mutation status, gene expression (96 targets), and alternative splicing (45 targets) within the same individual cell for 96 to 288 cells from 5 patients with different SF3B1 mutations. From the same patient sample, single cells with SF3B1 mutation generally exhibited increased alternative splicing for events identified from the bulk analysis, thus confirming the association of SF3B1 mutation with altered splicing at the single cell level. Different SF3B1 hotspot mutations within the HEAT repeat domains exhibited similar patterns of alternative splicing while a mutation outside of the repeat domain did not. Furthermore, we confirmed significant changes in gene expression between SF3B1 wild-type and mutant cells of target genes involved in the Notch pathway (NCOR2), cell cycle (CDKN2A, CCND1) and apoptosis (TXNIP). Consistent with these analyses, functional studies with overexpression of full-length mutated SF3B1 in a hematopoietic cell lines confirmed the modulation of these pathways by this putative CLL driver. Our high-resolution single cell analysis further uncovered 2 transcription factors strongly associated with SF3B1 mutation but not previously appreciated (KLF3 and KLF8). Our comprehensive transcriptomic analysis thus highlights SF3B1 mutation as an efficient mechanism by which a complex of changes relevant to CLL biology are generated that can contribute to disease progression. Disclosures Kipps: Pharmacyclics Abbvie Celgene Genentech Astra Zeneca Gilead Sciences: Other: Advisor. Li:Fluidigm: Employment. Livak:Fluidigm: Employment.

scholarly journals Full-length single-cell RNA-seq applied to a viral human cancer: applications to HPV expression and splicing analysis in HeLa S3 cells

GigaScience ◽  
2015 ◽  
Vol 4 (1) ◽  
Author(s):  
Liang Wu ◽  
Xiaolong Zhang ◽  
Zhikun Zhao ◽  
Ling Wang ◽  
Bo Li ◽  
...  
Keyword(s):  
Single Cell ◽  
Human Cancer ◽  
Full Length ◽  
Rna Seq ◽  
Hela S3 ◽  
Hela S3 Cells

scholarly journals Single-Cell RNA-Seq Reveals Heterogeneous lncRNA Expression in Xenografted Triple-Negative Breast Cancer Cells

Biology ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 987
Author(s):  
Holly R. Pinkney ◽  
Michael A. Black ◽  
Sarah D. Diermeier
Keyword(s):  
Breast Cancer ◽  
Single Cell ◽  
Cell Line ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Therapeutic Targets ◽  
Rna Seq ◽  
Tumour Heterogeneity ◽  
Lncrna Expression ◽  
Non Coding Rnas

Breast cancer is the most commonly diagnosed cancer in the world, with triple-negative breast cancer (TNBC) making up 12% of these diagnoses. TNBC tumours are highly heterogeneous in both inter-tumour and intra-tumour gene expression profiles, where they form subclonal populations of varying levels of aggressiveness. These aspects make it difficult to study and treat TNBC, requiring further research into tumour heterogeneity as well as potential therapeutic targets and biomarkers. Recently, it was discovered that the majority of the transcribed genome comprises non-coding RNAs, in particular long non-coding RNAs (lncRNAs). LncRNAs are transcripts of >200 nucleotides in length that do not encode a protein. They have been characterised as regulatory molecules and their expression can be associated with a malignant phenotype. We set out to explore TNBC tumour heterogeneity in vivo at a single cell level to investigate whether lncRNA expression varies across different cells within the tumour, even if cells are coming from the same cell line, and whether lncRNA expression is sufficient to define cellular subpopulations. We applied single-cell expression profiling due to its ability to capture expression signals of lncRNAs expressed in small subpopulations of cells. Overall, we observed most lncRNAs to be expressed at low, but detectable levels in TNBC xenografts, with a median of 25 lncRNAs detected per cell. LncRNA expression alone was insufficient to define a subpopulation of cells, and lncRNAs showed highly heterogeneous expression patterns, including ubiquitous expression, subpopulation-specific expression, and a hybrid pattern of lncRNAs expressed in several, but not all subpopulations. These findings reinforce that transcriptionally defined tumour cell subpopulations can be identified in cell-line derived xenografts, and uses single-cell RNA-seq (scRNA-seq) to detect and characterise lncRNA expression across these subpopulations in xenografted tumours. Future studies will aim to investigate the spatial distribution of lncRNAs within xenografts and patient tissues, and study the potential of subclone-specific lncRNAs as new therapeutic targets and/or biomarkers.

scholarly journals Single-Cell RNA-Seq Reveals Developmental Origins and Ontogenetic Stability of Neurexin Alternative Splicing Profiles

Cell Reports ◽  
2019 ◽  
Vol 27 (13) ◽  
pp. 3752-3759.e4 ◽  
Author(s):  
David Lukacsovich ◽  
Jochen Winterer ◽  
Lin Que ◽  
Wenshu Luo ◽  
Tamas Lukacsovich ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Single Cell ◽  
Developmental Origins ◽  
Rna Seq

scholarly journals Coverage-dependent bias creates the appearance of binary splicing in single cells

2019 ◽  
Author(s):  
Carlos F. Buen Abad Najar ◽  
Nir Yosef ◽  
Liana F. Lareau
Keyword(s):  
Alternative Splicing ◽  
Single Cell ◽  
Rna Splicing ◽  
Single Cells ◽  
Cell Types ◽  
Rna Seq ◽  
Unique Identity ◽  
Underlying Reality ◽  
Human And Mouse ◽  
Insight Into

Single cell RNA sequencing provides powerful insight into the factors that determine each cell’s unique identity, including variation in transcription and RNA splicing among diverse cell types. Previous studies led to the surprising observation that alternative splicing outcomes among single cells are highly variable and follow a bimodal pattern: a given cell consistently produces either one or the other isoform for a particular splicing choice, with few cells producing both isoforms. Here we show that this pattern arises almost entirely from technical limitations. We analyzed single cell alternative splicing in human and mouse single cell RNA-seq datasets, and modeled them with a probablistic simulator. Our simulations show that low gene expression and low capture efficiency distort the observed distribution of isoforms in single cells. This gives the appearance of a binary isoform distribution, even when the underlying reality is consistent with more than one isoform per cell. We show that accounting for the true amount of information recovered can produce biologically meaningful measurements of splicing in single cells.

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