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2018). This allowed the authors to identify transcriptional changes induced by repeated depolarization, a phenomenon believed to be critical for proper cortical function. To increase our resolution to identify neuronal activity programs, we used the published clustering to exclude any non-neuronal cells that were also captured by the experiment from our dataset. We sought to determine whether cNMF would identify the relatively modest activity programs (~60 genes with fold-change >= 2 and FDR We ran cNMF on neurons combined from all three exposure conditions and identified 20 GEPs, interpreting 14 as identity and six as activity programs (Figure 4—figure supplement 1). As we saw in the organoid data, the activity programs were co-expressed with many distinct and uncorrelated GEPs while the identity programs only overlapped in related cell types (Figure 4a–b). In addition, the identity programs were well separated by the published clusters while the activity programs were spread across multiple clusters (Figure 4—figure supplement 2). We thus used the published cluster annotations to label the identity GEPs. Identification of activity programs in neurons of the visual cortex. (a) Heatmap showing percent usage of all GEPs (rows) in all cells (columns). Identity GEPs are shown on top and activity GEPs are shown below. Cells are grouped by their maximum identity GEP and fit into columns of a fixed width for each identity GEP. (b) t-SNE plots of cells colored by maximum identity GEP usage (left) or by absolute usage of each activity GEP (right). (c) Box and whisker plot showing the percent usage of activity programs (rows) in cells classified according to their maximum identity GEP (columns) and stratified by the stimulus condition of the cells (hue). The central line represents the median, boxes represent the interquartile range, and whiskers represent the 5th and 95th quantile. (d) Scatter plot of z-scores of the superficial late response GEP in the primary dataset against the corresponding GEP in the Tasic et al. (2016) dataset. (e) Same as (d) but for the neurosecretory activity program. https://doi.org/10.7554/eLife.43803.019 Three activity programs were correlated with the stimulus, which indicates that they are induced by repeated depolarization (Figure 4c). One of these was induced at 1h and thus corresponds to an early response program (ERP). The others were primarily induced at 4h and thus correspond to late response programs (LRPs). These programs overlapped significantly with the sets of differentially expressed genes reported in Hrvatin et al. (2018) (p=8×10−34 for the ERP and genes induced at 1h; p=4×10−22, p=4×10−14 for the LRPs and genes induced at 4hs, one-sided Mann Whitney test).Intriguingly, one LRP was more induced in superficial cortical layers, while the other was more induced in deeper layers. This supports a recently proposed model where the ERP is predominantly shared across excitatory neurons, while LRPs vary more substantially across neuron subtypes (Hrvatin et al., 2018). It also illustrates cNMF’s sensitivity: in the initial report, only 64 and 53 genes were identified as differentially expressed in at least one excitatory cell type at 1h and 4hs (FC ≥2,

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Stimulus (Figure 5—figure supplement 1a - left). By contrast, there was significant variability between organoids in the Quadrato et al. (2017) data that was primarily associated with the bioreactors in which the organoids were grown (Figure 5—figure supplement 1b - left). This variability was discussed in the original manuscript and validated using immunohistochemistry, and thus represents true biological signal that we would hope for cNMF to discern.We also considered whether any GEPs could be attributed to just one or a small number of replicates which could suggest that they are not reproducible within the experiment. We therefore looked at what percentage of the aggregate usage of a GEP derived from cells in each replicate. We found that each GEP contributed to cells from multiple independent replicates in both datasets (Figure 5—figure supplement 1, right panels). No GEP derived more than 15% of its usage from a single replicate in the visual cortex data or more than 45% of its usage from a single replicate in the organoid data. Furthermore, each organoid GEP was the maximum contributing GEP for a cell in at least six distinct organoid replicates, and each visual cortex GEP was the maximum contributor for a cell in at least 10 distinct mouse replicates. This supports our conclusion that the inferred GEPs represent reproducible signals within the primary organoid and visual cortex datasets.We also analyzed a human pancreatic islet scRNA-Seq dataset where variability between four donors resulted in more substantial batch-effects to see how that would impact the behavior of cNMF (Baron et al., 2016). Applied to this dataset of 10,939 cells, cNMF identified 16 GEPs that corresponded well with the cell-type clusters described in the initial publication (Figure 5—figure supplement 2). Our application of cNMF failed to identify GEPs corresponding to a few cell-types described in Baron et al. (2016) (e.g. cells distinguished as delta and gamma cell-types were assigned the same GEP). However, many of the cell types that were missed by cNMF were only distinguished through iterative sub-clustering in the initial publication, which we did not attempt.Notably, we identified multiple GEPs for many cell-type clusters that corresponded to ‘donor of origin.’ For example, we identified separate GEPs corresponding to acinar cells derived from donors 1 and 3, and acinar cells derived from donors 2 and 4, and similarly for alpha, ductal, and stellate cells. One potential contributor to the batch-effect could be that donors 1 and 3 were male and donors 2 and 4 were female. Consistent with this, we noticed that among the genes that were most differentially expressed between donors 1 and 3 compared to donors 2 and 4 in alpha, beta, and acinar cells were XIST on the X chromosome and RPSY1 on the Y chromosome (linear regression F-test p-values−243 for for XIST and p-values−145 for RPSY1 for all 3 cell-types tested). But in general, the fact that cNMF is discerning multiple GEPs for the same cell-types suggests that technical sources of variation such as batch-effect can confound the identification of identity

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The house, compared with early/mid-February (e.g., because of working longer shifts as a key worker, increased working from home, furlough or redundancy)? Currently much more196549.3Currently a little more81920.5About the same116029.1Currently a little less250.6Currently much less170.4Compared to an average term-time week during early/mid-February 2020, (dog name) has spent... Much more time with the child(ren) in the household40571.3A little more time with the child(ren) in the household8615.1The same amount of time with the child(ren) in the household6611.6A little less time with the child(ren) in the household20.4Much less time the child(ren) in my household91.6 Table 2. The number of days/week and the maximum amount of time/day that the dog was reported to have been left alone in the home (during the last 7 days and during a week in early/mid-February 2020). Table 2. The number of days/week and the maximum amount of time/day that the dog was reported to have been left alone in the home (during the last 7 days and during a week in early/mid-February 2020). VariableBefore Lockdown (Early/Mid-February 2020)During Lockdown(The Last 7 Days)Relative Changen%Cumulative %n%Cumulative %Days left alone (for at least 5 min) in a week (n = 4276, χ2 = 2584.3, df = 7, p-value Not at all62214.514.5247357.857.84.01 day3839.023.583719.677.42.22 days46110.834.33949.286.60.93 days52412.346.51663.990.50.34 days46310.857.4751.892.30.25 days90121.178.41122.694.90.16 days2566.084.4210.595.40.17 days66615.6100.01984.6100.00.3Maximum duration left alone (n = 4272, χ2 = 2767.5, df = 6, p-value Not at all3367.97.9204147.847.86.1Less than 5 min330.88.61493.551.34.55–19 min842.010.62856.757.93.420–59 min2997.017.677418.176.12.61 or more hours but less than 3 h145434.051.689218.594.60.53 or more hours but less than 6 h169139.691.21683.998.50.16 or more hours3758.8100.0631.5100.00.2 Table 3. Daily walk frequency and total walk duration before and during lockdown period, and number of other dogs met (on walks or in an indoor space) during each period. Table 3. Daily walk frequency and total walk duration before and during lockdown period, and number of other dogs met (on walks or in an indoor space) during each period. VariableBefore Lockdown (Early/Mid-February 2020)During Lockdown(The Last 7 Days)Relative Changen%Cumulative %n%Cumulative %Number of walks (n = 4408, χ2 = 731.07, df = 4, p-value Not walked451.01.01072.42.42.41 per day149833.434.4221849.551.91.52 per day201244.979.4162336.288.10.83 per day67815.194.53828.596.70.64+ per day2475.5100.01503.3100.00.6Total duration of walks (n = 4480, χ2 = 70.16, df =

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