Non-genetic factors can cause individual cells to fluctuate substantially in gene expression levels over time. Yet it remains unclear whether these fluctuations can persist for much longer than the time for a single cell division. Current methods for measuring gene expression in single cells mostly rely on single time point measurements, making the time of a fluctuation of gene expression or cellular memory difficult to measure. Here, we report a method combining Luria and Delbrück's fluctuation analysis with population-based RNA sequencing (MemorySeq) for identifying genes transcriptome-wide whose fluctuations persist for several cell divisions. MemorySeq revealed multiple gene modules that express together in rare cells within otherwise homogeneous clonal populations. Further, we found that these rare cell subpopulations are associated with biologically distinct behaviors in multiple different cancer cell lines, for example, the ability to proliferate in the face of anti-cancer therapeutics. The identification of non-genetic, multigenerational fluctuations has the potential to reveal new forms of biological memory at the level of single cells and suggests that non-genetic heritability of cellular state may be a quantitative property. Main text:Cellular memory in biology, meaning the persistence of a cellular or organismal state over time, occurs over a wide range of timescales and can be induced by a variety of mechanisms. Genetic differences are one form of memory, encoding variation between organisms on multi-generational timescales. Within an organism, epigenetic mechanisms encode the differences between cell types in different tissues, with cells retaining memory of their state over a large number of cell divisions ( 1 ) . In contrast, recent measurements suggest that expression of many genes in single cells may have very little memory, displaying highly transient .

Molecular differences between individual cells can lead to dramatic differences in cell fate, such as death versus survival of cancer cells upon drug treatment. These originating differences remain largely hidden due to difficulties in determining precisely what variable molecular features lead to which cellular fates. Thus, we developed Rewind, a methodology that combines genetic barcoding with RNA FISH to directly capture rare cells that give rise to cellular behaviors of interest. Applied to BRAF V600E melanoma, we trace drug-resistant cell fates back to single-cell gene expression differences in their drug-naive precursors (initial frequency of ~1:1000–1:10,000 cells) and relative persistence of MAP-kinase signaling soon after drug treatment. Within this rare subpopulation, we uncover a rich substructure in which molecular differences between several distinct subpopulations predict future differences in phenotypic behavior, such as proliferative capacity of distinct resistant clones following drug treatment. Our results reveal hidden, rare-cell variability that underlies a range of latent phenotypic outcomes upon drug exposure.

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Molecular differences between individual cells can lead to dramatic differences in cell fate following an applied treatment, such as the difference between death versus survival of cancer cells upon receiving anti-cancer drugs. However, current strategies to retrospectively identify the cells that give rise to distinct rare behaviors and determine their distinguishing molecular characteristics remain limited. Here we describe Rewind, a methodology that combines genetic barcoding with an RNA-based readout to directly capture rare cells that give rise to cellular behaviors of interest, specifically the emergence of resistance to targeted cancer therapy. Using Rewind, we analyzed over 5 million cells to identify differences in gene expression and MAP-kinase signaling in single melanoma cells that mark a rare subpopulation of drug-naive cells (initial frequency of ~1:1000-1:10,000 cells) that ultimately gives rise to drug resistant clones. We further show that even within this rare subpopulation, molecular differences between single cells before the application of drug predict future differences in drug resistant behavior. Similarly, we show that treatments that modify the frequency of resistance can allow otherwise non-resistant cells in the drug-naive population to become resistant, and that these new populations are marked by the variable expression of distinct genes. Together, our results reveal the presence of cryptic variability that can underlie a range of distinct rare-cell phenotypic outcomes upon drug exposure. Applying Rewind to other rare biological phenomena, such as cancer metastasis, tissue regeneration, and stem cell reprogramming, may provide a means to map rare cellular states to the unique cellular fates to which they give rise.

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Splicing is the molecular process by which introns are removed from pre-mRNA and exons are joined together to form the sequence of the mature mRNA. Measuring the timing of splicing relative to the transcription of nascent RNA has yielded conflicting interpretations. Biochemical fractionation suggests that RNA is spliced primarily during the process of transcription, but imaging of nascent RNA suggests that splicing happens after the process of transcription has been completed. We use single molecule RNA FISH together with expansion microscopy to measure the spatial distribution of nascent and partially spliced transcripts in mammalian cells, allowing us to infer the delay between when an intron is transcribed and when it is spliced out of a pre-mRNA. We show that 4 out of 4 genes we interrogated exhibit some post-transcriptional splicing, and that introns can be spliced in any order. We also show that completely synthesized RNA move slowly through a transcription site proximal zone while they undergo additional splicing and potentially other processing after transcription is completed. In addition, upon leaving this zone, some genes' transcripts localize to speckles during the process of splicing but some appear to traffic freely through the nucleus without localizing to any other nuclear compartment. Taken together, our observations suggest that the regulation of the timing and localization of splicing is specific to individual introns, as opposed to the previously surmised immediate excision of introns after transcription.

This work demonstrates an open-source hardware and software platform for monitoring the performance of buildings, called Elemental, that is designed to provide data on indoor environmental quality, energy usage, HVAC operation, and other factors to its users. It combines: (i) custom printed circuit boards (PCBs) with RFM69 frequency shift keying (FSK) radio frequency (RF) transceivers for wireless sensors, control nodes, and USB gateway, (ii) a Raspberry Pi 3B with custom firmware acting as either a centralized or distributed backhaul, and (iii) a custom dockerized application for the backend called Brood that serves as the director software managing message brokering via Message Queuing Telemetry Transport (MQTT) protocol using VerneMQ, database storage using InfluxDB, and data visualization using Grafana. The platform is built around the idea of a private, secure, and open technology for the built environment. Among its many applications, the platform allows occupants to investigate anomalies in energy usage, environmental quality, and thermal performance via a comprehensive dashboard with rich querying capabilities. It also includes multiple frontends to view and analyze building activity data, which can be used directly in building controls or to provide recommendations on how to increase operational efficiency or improve operating conditions. Here, we demonstrate three distinct applications of the Elemental platform, including: (1) deployment in a research lab for long-term data collection and automated analysis, (2) use as a full-home energy and environmental monitoring solution, and (3) fault and anomaly detection and diagnostics of individual building systems at the zone-level. Through these applications we demonstrate that the platform allows easy and virtually unlimited datalogging, monitoring, and analysis of real-time sensor data with low setup costs. Low-power sensor nodes placed in abundance in a building can also provide precise and immediate fault-detection, allowing for tuning equipment for more efficient operation and faster maintenance during the lifetime of the building.

Pluripotency can be induced in somatic cells by the expression of the four Yamanaka factors OCT4, KLF4, SOX2, and MYC. However, even in homogeneous conditions, usually only a rare subset of cells admit reprogramming, and the molecular characteristics of this subset remain unknown. Here, we apply retrospective clone tracing to identify and characterize the individual human fibroblast cells that are primed for reprogramming. These fibroblasts showed markers of increased cell cycle speed and decreased fibroblast activation. Knockdown of a fibroblast activation factor identified by our analysis led to increased reprogramming efficiency, identifying it as a barrier to reprogramming. Changing the frequency of reprogramming by inhibiting the activity of LSD1 led to an enlarging of the pool of cells that were primed for reprogramming. Our results show that even homogeneous cell populations can exhibit heritable molecular variability that can dictate whether individual rare cells will reprogram or not.