BASIC: B-cell receptor (BCR) assembly from single cell RNA-seq
ABSTRACT: The B-cell receptor (BCR) enables individual B cells to identify diverse antigens, including bacterial and viral proteins. While advances in RNA-seq have enabled high throughput profiling of transcript expression in single cells, the unique task of assembling the full-length heavy and light chain sequences from single cell RNA-sequencing (scRNA-seq) in B cells has been largely unstudied. We developed a new software tool, BASIC, which allows investigators to use scRNA-seq for assembling BCR sequences at single cell level. To demonstrate the utility of our software, we subjected single B cells from a human donor to scRNA-seq, assembled the full-length heavy and the light chains, and experimentally confirmed these results by using single cell primer based nested PCRs and Sanger sequencing.
Project description:Motivation: The B-cell receptor (BCR) performs essential functions for the adaptive immune system including recognition of pathogen-derived antigens. The vast repertoire and adaptive variation of BCR sequences due to V(D)J recombination and somatic hypermutation (SHM) necessitates single-cell characterization of BCR sequences. Single-cell RNA sequencing (scRNA-seq) presents the opportunity for simultaneous capture of paired BCR heavy and light chains and the transcriptomic signature. Results: We developed VDJPuzzle, a novel bioinformatic tool that reconstructs productive, full-length B-cell receptor sequences of both heavy and light chains. VDJPuzzle successfully reconstructed BCRs from 98.3% (n=117) human and 96.5% (n=200) murine B cells. The reconstructed BCRs were successfully validated with single-cell Sanger sequencing. Availability: VDJPuzzle is available at https://bitbucket.org/kirbyvisp/vdjpuzzle2
Project description:Pre-B cell receptor (pre-BCR) signals initiate immunoglobulin light (Igl) chain gene assembly leading to RAG-mediated DNA double-strand breaks (DSBs). These signals also promote cell cycle entry, which could cause aberrant DSB repair and genome instability in pre-B cells. Here we show that RAG DSBs inhibit pre-BCR signals through the ATM- and NF-κB2-dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor to prevent the pre-BCR from inducing additional Igl chain gene rearrangements and driving pre-B cells with RAG DSBs into cycle. We propose that pre-B cells toggle between pre-BCR signals and this RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes. Overall design: Three independent IL-7 cultures for each genotype (Rag1-/-:μIgH:Bcl2, Art-/-:μIgH:Bcl2 and Art-/-:Nfkb2-/-:μIgH:Bcl2) were withdrawn from IL-7 for 2 days. RNA was isolated using RNeasy (Qiagen). Gene expression profiling was performed using Illumina MouseRef-8 expression microarrays.
Project description:Pre-B cell receptor (pre-BCR) signals initiate immunoglobulin light (Igl) chain gene assembly leading to RAG-mediated DNA double-strand breaks (DSBs). These signals also promote cell cycle entry, which could cause aberrant DSB repair and genome instability in pre-B cells. Here we show that RAG DSBs inhibit pre-BCR signals through the ATM- and NF-κB2-dependent induction of SPIC, a hematopoietic-specific transcriptional repressor. SPIC inhibits expression of the SYK tyrosine kinase and BLNK adaptor to prevent the pre-BCR from inducing additional Igl chain gene rearrangements and driving pre-B cells with RAG DSBs into cycle. We propose that pre-B cells toggle between pre-BCR signals and this RAG DSB-dependent checkpoint to maintain genome stability while iteratively assembling Igl chain genes. Three independent IL-7 cultures for each genotype (Rag1-/-:μIgH:Bcl2, Art-/-:μIgH:Bcl2 and Art-/-:Nfkb2-/-:μIgH:Bcl2) were withdrawn from IL-7 for 2 days. RNA was isolated using RNeasy (Qiagen). Gene expression profiling was performed using Illumina MouseRef-8 expression microarrays.
2016-01-13 | E-GEOD-67854 | ArrayExpress
Project description:Paired Heavy and Light Chain Immunoglobulin Reconstruction in Single-Cell RNA-Seq Data
Project description:Single-cell RNA sequencing (scRNA-seq) methods generate sparse gene expression profiles for thousands of single cells in a single experiment. The information in these profiles is sufficient to classify cell types by distinct expression patterns but the high complexity of scRNA-seq libraries prevents full characterization of transcriptomes from individual cells. To generate more focused gene expression information from scRNA-seq libraries, we developed a strategy to physically recover the DNA molecules comprising transcriptome subsets, enabling deeper interrogation of the isolated molecules by another round of DNA sequencing. We applied the method in both a cell-centric and gene-centric mode to isolate mRNA fragments from scRNA-seq libraries. Overall design: targeted resequencing and original untargeted datasets of various scRNA-Seq libraries from the 10x genomics and wafergen platform
Project description:We report that B-1a cells develop in a surrogate light chain independent context. As a consequence, the precursor B-1a cell population avoids a pre-BCR positive selection stage. To confirm that the B-1a cells generated in this manner repersent a bonafide B-1a cell compartment, we did NGS on BCR rearrangements to assess the repertoire diversity. We find that as a whoile, B-1a cell repertoire that develop in Igll1 kncokout mice are similar compared to wild-type. This supports our findings that B-1a cells develop properly in the absence of surrogate light chain. Overall design: Peritoneal cavity B-1a cells were sorted from 6-8 week old mice, DNA was extracted and rearranged heavy chains were amplified by PCR. These PCR products were then process to form DNA libraries that were compatible with Illumina NGS.
Project description:During a germinal center (GC) response, B cells diversify their immunoglobulin (Ig) genes by somatic hyper-mutation (SHM) and undergo clonal expansion and positive selection thereby enabling the generation of higher affinity antibodies. We have analyzed the genomic states underlying GC B cell dynamics by single cell RNA-Seq. Profiling of antigen specific GC B cells during the peak of the response, revealed four distinctive genomic states characterized by antigen presentation, apoptotic, mitochondrial and mitotic gene expression modules. Intersection of genomic states and Ig heavy-chain (Igh) class-switch trajectory suggested that mitochondrial machinery is utilized to support class-switch recombination (CSR). Furthermore, by analyzing the transcriptomes of B cells with varying affinity BCR sequences that assembled from single-cell RNA-seq data through a novel algorithm, we show that high affinity GC B cells manifest enhanced mitotic and BCR signaling transduction, but compromised antigen processing and presentation gene expression modules. Thus, we are developing a comprehensive framework of the genomic states and molecular pathways underlying GC B cell dynamics. Overall design: C56BL/6J (Jax 664) mice were immunized intraperitoneally with 100 μg NP(23)-KLH (Biosearch Technologies) mixed with 50% (v/v) Alum (Thermo Scientific) and 1 μg LPS (Sigma). Splenocyte suspensions were prepared in MACS buffer (pH 7.4 PBS plus 1% FBS and 5 mM EDTA) on day 13 post immunization and blocked with 25 μg/ml 2.4G2 (BD) for 15 min on ice. Then cells were labeled with 2 μg/ml biotin anti-CD3, 1 μg/ml biotin anti-CD4, 1 μg/ml biotin anti-CD8, 2.5 μg/ml biotin anti-CD11C and 2.5 μg/ml biotin anti-IgD for 25 min at 4°C. After washing four times, cells were further labeled with anti-biotin beads (Miltenyi Biotec) for 20 min at 4°C. The magnetic columns were used to deplete non-GC B cells according to standard protocols (Miltenyi Biotec). The effluent cells were further labeled with NP-PE, B220, Fas and GL7 antibodies (Supplementary Table 1) for 30 min at 4 °C. GC B cells were sorted as 7AAD–B220+Fas+GL7hi with FACSAria II (BD) with 70 μm nozzle. Single GC B cells were prepared using the C1TM Single-Cell Auto Prep System (Fluidigm, San Fransisco, CA), according to the manufacturer’s instructions. 182 scRNA Seq libraries were sequenced per HiSeq 2500 gel with 75bp paired-end sequencing.
Project description:Proliferating cells known as neoblasts include pluripotent stem cells (PSCs) capable of sustaining tissue homeostasis and regeneration of lost body parts in planarians. However, the lack of markers to prospectively identify and isolate these adult PSCs has significantly hampered their characterization. We used single-cell RNA sequencing (scRNA-seq) and single cell transplantation to address this long-standing issue. Large-scale scRNA-seq of fluorescently sorted neoblasts unveiled a novel subtype of neoblast (Nb2) characterized by high levels of PIWI-1 mRNA and protein, and marked by a conserved cell-surface protein coding gene, tetraspanin 1 (tspan1). tspan1-positive cells survived sub-lethal irradiation, underwent clonal expansion to repopulate whole animals, and when purified with an anti-TSPAN-1 antibody rescued with high efficiency the viability of lethally irradiated animals after single-cell transplantation. Our work demonstrates the first prospective isolation of an adult PSC, bridges a conceptual dichotomy between operationally and molecularly defined neoblasts, and sheds light on the mechanisms governing in vivo pluripotency and the source of regeneration in planarians. Overall design: 1) Examine gene expression profiles of sorted 80 X1 and 80 Xins cell at single cell level using the plate based single cell RNA-seq platform. Two batch of (40 X1+40 Xins) cells were sorted into individual 96-well plates, and libraries were made by ClonTech v4 full reactions, and quarter-size reactions, respectively. 2) Examine gene expression profiles of sorted X1 cells, and X1+X2 cells survived in 7-day irradiation by 10X scRNA-Seq platform.
Project description:Expression of stereotyped B cell receptors (BCR), i.e. non-random combinations of immunoglobulin heavy-chain variable (IGHV) genes, complementarity-determining region-3 (HCDR3), and IGV light chains, identifies discrete clusters and represents a peculiar feature of chronic lymphocytic leukemia (CLL). Expression of IGHV3-23 characterized a CLL subset with peculiar molecular and clinical features. Overall design: Two-condition experiment, Mutated IGVH3-23 vs. Mutated non-IGVH3-23. Biological replicates: 22 control replicates, 5 Mutated IGVH3-23 replicates. Refrence design
Project description:In this study, we assess technical differences between commonly used single-cell RNA-Sequencing (scRNA-Seq) methods. We perform scRNA-seq on a homogenous population of mouse embryonic stem cells along with two kinds of control spike-in molecules to assess sensitivity and accuracy of these specific methods. In this dataset, we perform SMARTer method on Fluidigm C1 system and generate single-cell libraries using Nextera XT kit