Recent molecular studies have shown that, even when derived from a seemingly homogenous population, individual cells can exhibit substantial differences in gene expression, protein levels and phenotypic output1,2,3,4,5, with important functional consequences4,5. Existing studies of cellular heterogeneity, however, have typically measured only a few pre-selected RNAs1,2 or proteins5,6 simultaneously, because genomic profiling methods3 could not be applied to single cells until very recently7,8,9,10. Here we use single-cell RNA sequencing to investigate heterogeneity in the response of mouse bone-marrow-derived dendritic cells (BMDCs) to lipopolysaccharide. We find extensive, and previously unobserved, bimodal variation in messenger RNA abundance and splicing patterns, which we validate by RNA-fluorescence in situ hybridization for select transcripts. In particular, hundreds of key immune genes are bimodally expressed across cells, surprisingly even for genes that are very highly expressed at the population average. Moreover, splicing patterns demonstrate previously unobserved levels of heterogeneity between cells. Some of the observed bimodality can be attributed to closely related, yet distinct, known maturity states of BMDCs; other portions reflect differences in the usage of key regulatory circuits. For example, we identify a module of 137 highly variable, yet co-regulated, antiviral response genes. Using cells from knockout mice, we show that variability in this module may be propagated through an interferon feedback circuit, involving the transcriptional regulators Stat2 and Irf7. Our study demonstrates the power and promise of single-cell genomics in uncovering functional diversity between cells and in deciphering cell states and circuits.
Access optionsAccess options
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Gene Expression Omnibus
Data have been deposited in GEO under accession number GSE41265.
We thank N. Chevrier, C. Villani, M. Jovanovic, M. Bray and J. Shuga for scientific discussions; N. Friedman and E. Lander for comments on the manuscript; B. Tilton, T. Rogers and M. Tam for assistance with cell sorting; J. Bochicchio, E. Shefler and C. Guiducci for project management; the Broad Genomics Platform for all sequencing work; K. Fitzgerald for the Irf7−/− bone marrow; and L. Gaffney for help with artwork. Work was supported by a National Institutes of Health (NIH) Postdoctoral Fellowship (1F32HD075541-01, to R.S.), a Charles H. Hood Foundation Postdoctoral Fellowship (to A. Goren), an NIH grant (U54 AI057159, to N.H.), an NIH New Innovator Award (DP2 OD002230, to N.H.), an NIH CEGS Award (1P50HG006193-01, to H.P., A.R. and N.H.), NIH Pioneer Awards (5DP1OD003893-03 to H.P., DP1OD003958-01 to A.R.), the Broad Institute (to H.P. and A.R.), HHMI (to A.R.), and the Klarman Cell Observatory at the Broad Institute (to A.R.).
This zipped file contains Supplementary Tables 1-7. Supplementary Table 1 shows sequencing metrics for single cell, population, and molecularly barcoded RNA-seq libraries. Supplementary Table 2 shows transcript per million (TPM) levels for all UCSC genes (rows) for 18 single cells and 3 population replicates (columns), along with annotation of which genes are 'Housekeeping' and which are 'LPS Response'. Supplementary Table 3 shows single cell variability measures for 523 highly expressed (population average) genes, along with annotation of which genes are 'Housekeeping' and which are 'LPS Response'. Supplementary Table 4 shows percent spliced in (PSI) estimates for all genes that are very highly expressed (TPM>250) in at least one single cell, only PSI estimates for the highly expressing cells were used to generate Figure 3b (see Supplementary Information file). Supplementary Table 5 shows clustering assignments and principal component scores for 633 genes induced in response to LPS stimulation. Supplementary Table 6 contains gene list and PCR primer pairs used for the Fluidigm single cell qPCR codeset. Supplementary Table 7 contains TPM estimates and unique molecular identifier counts for each gene in the 3 libraries prepared using the modified SMARTer protocol.
About this article
Current Bioinformatics (2019)