Massively parallel simultaneous profiling of the transcriptomic and epigenomic landscape at single cell resolution
Shamoni Maheshwari, Sharmila Chatterjee, Jerald Sapida, Joe Shuga, Li Wang, Yiming Kang, Brett Olsen, Vijay Kumar, Corey Nemec, Martin Sauzade, Maengseok Song, Catie McConnell, Jason Bell, Jean Wang & Kamila Belhocine 10x Genomics, Inc., Pleasanton, CA
Introduction Parsing cellular heterogeneity within a lymphoma • We present here a single cell high-throughput assay to simultaneously profile 3’ gene expression (GEX) and open chromatin (via ATAC-seq) We generated joint GEX and ATAC data from 9158 single cells from a CD20+ diffuse small cell lymphoma sample. Using gene expression from the same nucleus. markers, we annotated the immune cell types present and could classify this tumor as a gastric MALT lymphoma (data not shown). We used • Such simultaneous profiling allows for finer resolution of cell types both in terminally differentiated cells and cells on a developmental trajectory. two orthogonal lines of evidence to differentiate between tumor and normal B cells: i) mapping mutation load using SNPs from a TCGA B cell • This assay has the potential to reveal the complex interplay between regulatory elements, open chromatin and gene expression. lymphoma project and ii) the BANK1-CD40 axis to identify the hyper-activated tumor B cells. Workflow A. GEX-derived tsne
• The assay input consists of a single nuclei suspension that undergoes transposition in bulk. CD20 SNVS alternate alelle We used publicly replicating tumor B cells reference alelle Weavailable used publicly mutation • Transposed nuclei are partitioned using a microfluidic chip along with Gel Beads that contain barcoded oligonucleotides and enzymes that B cells no data availabledata from mutation the dataTCGA-DLBC from the project enable the simultaneous conversion of polyadenylated mRNA and open chromatin into barcoded cDNA and ATAC fragments, respectively. TCGA-DLBCon Diuse projectLarge onB Diffuse cell Lymphoma Large B normal B cells • After partitioning a standard NGS workflow yields two libraries: a barcoded 3’ GEX library and a barcoded ATAC library. replicating celland Lymphoma filtered it and to T cells filteredretain it onlyto retain 279 SNVs • These libraries can be sequenced on a variety of Illumina sequencers. onlythat 279 were SNVs predicted that wereto have predicted a deleterious to • The sequencing reads are processed via Cell Ranger, a turnkey software solution that performs barcoded read alignment, cell barcode havephenotype a deleterious and phenotypepresent in and the dbSNP database. identification, and secondary analyses like dimensionality reduction, clustering, and differential analyses. monocytes present in the dbSNP database. • Furthermore, the GEX and ATAC data can be analyzed using Loupe Browser, allowing the user to explore the data in an intuitive manner. T cells B. ATAC-derived tsne
BANK1 (B cell scaold Collect GEM Post GEM-RT ATAC Single Cell BANK1 CD40 monocytes protein with ankyrin Incubation Cleanup SI-PCR Pre-amplification ATAC Library BANK1repeats (B cell 1) modulatesscaffold protein with ankyrin 10x Barcoded normal B cells B-cell antigen Gel Beads repeatsreceptor 1) modulates (BCR)- cDNA Amplification B-cellinduced antigen calcium Gene receptormobilization (BCR)- and Oil in Well Expression inducedweakens calcium CD40- mobilizationmediated andAkt Fragmentation weakensactivation CD40- to End Repair & Ligation mediatedprevent Akt B cell activationhyper-activation to [1]. Transposed tumor B cells prevent B cell Nuclei hyper-activation [1]. T cells Enzymes SI-PCR
Transposition of Single Nuclei 10x Barcoded Accessible 10x Barcoded Nuclei in bulk GEMs DNA Fragments + DNA + RNA Single Cell Barcoded mRNA Gene Expression Library Identifying a tumor-specific signaling pathway Dysregulation of the JAK-STAT pathway is frequently observed in many primary human tumors. Differential gene expression analysis Sample prep GEM generation Library construction Sequencing Data processing Data visualization revealed over-expression of the IL4 receptor (IL4R) in the tumor B cells (A), and furthermore, we found that the STAT6 binding motif is accessible specifically in the tumor B cells (B).
Prepare nuclei A B IL4 Stat3 Stat6 suspension SignalSignal transducer transducer
normal B tumor B P andand activator activator of of P Jak3 Expression transcriptiontranscription (STAT) (STAT) FCRL5 Jak1 P proteinsproteins are are critical critical mediators of cytokine GRHPR P Stat6 mediators of cytokine signaling. However, P Stat3 signaling. However, MBD4 STATsSTATs are are latent latent cytoplasmiccytoplasmic proteins proteins A sensitive and scalable solution MIR155HG makingmaking expression expression
a poor proxy for
P a poor proxy for P Stat3
• Shown in panel A are the results of a 5000 cell PBMC experiment where the ATAC and GEX libraries were sequenced to a depth of ~65k and FCRL3 Stat6 function. Stat3 Stat6 function. 57k raw read pairs per cell, respectively. The partitions that contain nuclei (in yellow) have strong ATAC signal (measured as high mapping P P IL4R IL4 quality fragments on X axis) and GEX signal (measured as high mapping quality transcriptomic UMI counts on Y axis) and are well separate UponUpon Jak-mediated Jak-mediated RASGRF1 Stat3 MOTIF Stat6 MOTIF phosphorylationphosphorylation an an activated STAT from the noise (in black) due to empty partitions. P activated STAT 1.0 P Jak3 translocates to the IGF2BP3 Accessibility translocates to the Jak1 nucleus and binds to • The assay allows for profiling between 500 to 10,000 single nuclei in a single chip well. Shown in panel B are the results of loading ~1.5k cells P nucleus and binds to QSOX2 itsits DNA-recognition DNA-recognition 0.5 P Stat6 motifs,motifs, in inthe the that are a mixture of human GM12878 and mouse 3T3 nuclei. Note that most barcodes either contain exclusively human reads (yellow, 567 cells) P Stat3 PDGFD promoterspromoters of of or exclusively mouse reads (green, 935 cells) and 13 observed doublets (blue). Based on the number of observed human-mouse doublets, 0 cytokine-induciblecytokine-inducible IGLC2 genes.genes. Among Among the the
we estimate the total doublet rate at approximately 1% in this experiment. sevenseven STAT STAT proteins, proteins,
P P TIMP1 Stat3
−0.5 Stat6 STAT6STAT6 is isactivated activated by by Stat3 Stat6 • Shown in panel C is a comparison of nuclei from an embryonic mouse brain (E18) sample split and run on Chromium Single Cell Gene P P FN1 IL-4IL-4 and and IL-13 IL-13 [2]. [2]. −1.0 Expression v3, Chromium Single Cell ATAC and Chromium Single Cell ATAC + Gene Expression. Here we show that the sensitivity of the joint assay IL4 GBP5 Bcl-2 IgE is comparable to the single assays (on transposed nuclei) as a function of average sequencing depth per cell. STAT6STAT6 activation activation is is UTRN P P Jak3
Expression knownknown to to promote promote Jak1 immunoglobulin GBP1 P immunoglobulin A.5k PBMC B. 1.5k barnyard C. 1k embryonic mouse brain (E18) classclass switching switching to to IgE IgE P Stat6 andand prevention prevention of of LMO4 P Stat3 apoptosisapoptosis through through