Unknown,Transcriptomics,Genomics,Proteomics

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MiSeq paired-end sequencing of HEK293T cells stably expressing a Cas9/gRNA-dependent editable reporter vector to test novel nucleobase-editing techniques

ABSTRACT: The goal of these experiments were to test the on-target and target-adjacent editing efficiencies of different single-nucleobase editing systems. Previous studies have shown that tethering DNA mutating enzymes to Cas9-nickase-UGI complexes results in editing of chromosomal DNA. However, these editing events encompass undesirable target-adjacent nucleobase edits. Here, we characterize a novel approach that reduces the frequency of target-adjacent editing while maintaining a high level of on-target editing.

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Homo sapiens

SUBMITTER: Reuben Harris

PROVIDER: E-MTAB-8742 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Purpose: RNA editing by ADAR1 is essential for hematopoietic development. The goals of this study were firstly, to identify ADAR1-specific RNA-editing sites by indentifying A-to-I (G) RNA editing sites in wild type mice that were not edited or reduced in editing frequency in ADAR1 deficient murine erythroid cells. Secondly, to determine the transcription consequence of an absence of ADAR1-mediated A-to-I editing. Methods: Total RNA from E14.5 fetal liver of embryos with an erythroid restricted deletion of ADAR1 (KO) and littermate controls (WT), in duplicate. cDNA libraries were prepared and RNA sequenced using Illumina HiSeq2000. The sequence reads that passed quality filters were analyzed at the transcript level with TopHat followed by Cufflinks. qRTâPCR validation was performed using SYBR Green assays. A-to-I (G) RNA editing sites were identified as previously described by Ramaswami G. et al., Nature Methods, 2012 using BurrowsâWheeler Aligner (BWA) followed by ANOVA (ANOVA). RNA editing sites were confirmed by Sanger sequencing. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 14,484 transcripts in the fetal livers of WT and ADAR1E861A mice with BWA. RNA-seq data had a goodness of fit (R2) of >0.7, p<0.0001 between biological duplicates per genotype. Clusters of hyper-editing were onserved in long, unannotated 3'UTRs of erythroid specific transcripts. A profound upregulation of interferon stimulated genes were found to be massively upregulated (up to 5 log2FC) in KO fetal liver compared to WT. 11.332 (6,894 novel) A-to-I RNA editing sites were identified when assessing mismatches in RNA-seq data. Conclusions: Our study represents the first detailed analysis of erythroid transcriptomes and A-to-I RNA editing sites, with biologic replicates, generated by RNA-seq technology. A-to-I RNA editing is the essential function of ADAR1 and is required to prevent sensing of endogenous transcripts, likely via a RIG-I like receptor mediated axis. Fetal liver mRNA profiles of E14.5 wild type (WT) and ADAR Epor-Cre knock out mice were generated by deep sequencing, in duplicate using Illumina HiSeq 2000.

Project description:The discovery of hepatitis C virus (HCV) in 1989 revealed the virus as the etiology of 40%-90% of the “essential” mixed cryoglobulinemia, where immune complex forms deposit called cryoprecipitate at temperatures below 37 °C. Cryoprecipitate constitutes monoclonal IgM and polyclonal IgG, some of which has reactivity against HCV core and NS3 epitopes. Resultant immune complex is considered entrapped on microvascular endothelium via C1q receptor, leading to complement activation and organ injury presenting predominantly as dermopathy, peripheral neuropathy and nephropathy. However, currently little is known on whether auto-reactive, cold-precipitating IgG components enriched in cryoprecipitate may play some role on the deposition of immune complex and subsequent complement-mediated injury of specific organs. Recently, with the advent of high-throughput immune repertoire sequencing and mass spectrometry, technical feasibility is growing to delineate antibodies of interest and their sequences directly from serum. To date, vast majority of studies actually utilized the antigen column for affinity purification of antibodies of interest, although this strategy is not applicable to disease entity with unknown antigen involvement. In such cases, disease-specific and organ-specific immune deposits may be a good alternative source of etiological antibodies. Herein, targeting HCV cryoglobulinemic vasculitis as a model, we conducted a proof-of-concept study aiming at characterizing the IgG components most prone to cryoprecipitation. To this end, we longitudinally studied one patient with cryoglobulinemic vasculitis with chronic HCV infection. After obtaining informed consent, cryoprecipitate and supernatant were separated from peripheral blood sample. Fab fragments from Protein G-purified IgG were recovered after papain digestion for isobaric tags for relative and absolute quantification (iTRAQ)-based quantitative proteomics. Simultaneously, total RNA was isolated from peripheral blood, and immunoglobulin heavy chain variable region (IGHV) was PCR-amplified with unique molecular identifier (UMI) strategy to construct a personal IGHV sequence library of immunoglobulin variable region. Sequencing output from MiSeq was bioinformatically converted into mass spectrometry database. Search was performed using MaxQuant software.

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MiSeq paired-end sequencing of HEK293T cells stably expressing a Cas9/gRNA-dependent editable reporter vector to test novel nucleobase-editing techniques

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