Nature - USA (2019-07-18)

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nature research | reporting summary

October 2018

Field-specific reporting

Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences

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Life sciences study design

All studies must disclose on these points even when the disclosure is negative.

Sample size We performed scSLAM-seq on 107 single murine fibroblast cells and in parallel analyzed global transcriptional changes of a matched larger

(1x10^5) population of cells using SLAM-seq. We aimed to sequence about 50 cells per condition to get reproducible gene expression data

(from at least 5 cells that express the gene) and analyze transcriptional activity of genes which were transcribed in only 10% of cells.

We performed a 10x Chromium droplet-based scRNA-seq run to compare droplet-based scRNA-seq data with our scSLAM-seq approach. The

respective run provided us with hundreds of uninfected (n=793) and MCMV-infected (n=353). This was clearly sufficient to compare to the 49

CMV-infected and 45 uninfected cells.

Data exclusions For scSLAM-seq, we filtered out cells with less 2,500 quantified genes (TPM>1) (Extended Data Fig. 1a). Using a cut-off of 2,500 quantified

genes per single cell is a well-established cut-off in the field of scRNA-seq and simply ensures that only cells with decent coverage rates of

their transcriptome are included into the subsequent analyses.

Replication During the establishment of scSLAM-seq, we first prepared scSLAM-seq libraries from 50, 5 and 1 cell. Only once this was succesful, we

analzyed two biological replicates (Extended Data Fig. 1a). These reproduced and confirmed the findings from both the smaller number of

cells, cell pools and bulk sequencing data.

Randomization No randomization was required for this study as samples only comprised uninfected and infected cells.

Blinding Investigators were not blinded during data collection and analysis as this would not have been possible during sample preparation, data

collection and analysis. Furthermore, blinding was not relevant to this study. Only scSLAM-seq and the analysis of new RNA enables us to

differentiate the infected from the uninfected cells.

Reporting for specific materials, systems and methods

We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material,

system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Materials & experimental systems

n/a Involved in the study

Antibodies

Eukaryotic cell lines

Palaeontology

Animals and other organisms

Human research participants

Clinical data

Methods

n/a Involved in the study

ChIP-seq

Flow cytometry

MRI-based neuroimaging

Eukaryotic cell lines

Policy information about cell lines

Cell line source(s) Murine NIH-3T3 fibroblasts were obtained from ATCC (ATCC® CRL-1658).

Authentication Not authenticated but can be deduced from RNA-seq data.

Mycoplasma contamination Cell culture was negatively tested for Mycoplasma contamination by PCR.

Commonly misidentified lines

(See ICLAC register)

None of the cell lines used in this study is listed in the ICLAC register.