High-throughput RNA sequencing of paraformaldehyde-fixed single cells

  1. Hoang Van Phan
  2. Michiel van Gent
  3. Nir Drayman
  4. Anindita Basu
  5. Michaela U. Gack
  6. Savaş Tay

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Abstract

Single-cell transcriptomic studies that require intracellular protein staining, rare cell sorting, or inactivation of infectious pathogens are severely limited. This is because current high-throughput single-cell RNA sequencing methods are either incompatible with or necessitate laborious sample preprocessing for paraformaldehyde treatment, a common tissue and cell fixation and preservation technique. Here we present FD-seq (Fixed Droplet RNA sequencing), a high-throughput method for droplet-based RNA sequencing of paraformaldehyde-fixed, permeabilized and sorted single cells. We show that FD-seq preserves the RNA integrity and relative gene expression levels after fixation and permeabilization. Furthermore, FD-seq can detect a higher number of genes and transcripts than methanol fixation. We first apply FD-seq to analyze a rare subpopulation of cells supporting lytic reactivation of the human tumor virus KSHV, and identify TMEM119 as a potential host factor that mediates viral reactivation. Second, we find that infection with the human betacoronavirus OC43 leads to upregulation of pro-inflammatory pathways in cells that are exposed to the virus but fail to express high levels of viral genes. FD-seq thus enables integrating phenotypic with transcriptomic information in rare cell subpopulations, and preserving and inactivating pathogenic samples.

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  1. Version published to 10.1038/s41467-021-25871-2
  2. Version published to 10.1101/2020.09.17.302232v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2020.09.17.302232: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    RandomizationTo calculate the technical variance, we normalized the UMI count by converting it to UMIs per million, then randomly chose 400 cells from each fixation method and used the 1,000 most highly expressed non-mitochondrial genes to calculate the mean and squared coefficient of variation.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    After washing and blocking the cells in PBS supplemented with 4% BSA, staining was performed with an anti-KSHV K8.1 antibody (sc-65446, Santa Cruz, 1:50 dilution) and an Alexa Fluor 488-conjugated goat-anti-mouse secondary antibody (A10667, Life Technologies, 1:4000 dilution) in PBS/1% BSA supplemented with RNase inhibitor, murine (NEB).
    anti-KSHV K8.1
    suggested: (Advanced Biotechnologies Cat# 13-213-100, RRID:AB_1929220)
    goat-anti-mouse
    suggested: (Electron Microscopy Sciences Cat# 815.022, RRID:AB_2629849)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: HEK293T (ATCC) cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v
    HEK293T
    suggested: None
    HEK293T.rKSHV.219 cells were generated by infecting HEK293T cells with rKSHV.21924, and selecting with 1 μg/mL puromycin.
    HEK293T.rKSHV.219
    suggested: None
    The KSHV-positive human PEL cell line BC3 was cultured in Roswell Park Memorial Institute (RPMI) supplemented with 20% (v/v)
    BC3
    suggested: None
    Infection of A549 cells: OC43 (ATCC) were grown and titrated on A549 cells.
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    HEK293T.rKSHV219 cells were reverse transfected with 0.8 μg plasmid DNA using Lipofectamine2000 (Life Technologies) following the manufacturer’s instructions and seeded in 12 or 24-well plates for analysis by live-cell imaging or qRT-PCR as indicated.
    HEK293T.rKSHV219
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    The 3T3 cells (p65−/− 3T3 mouse embryonic fibroblast cells expressing p65-DsRed and H2B-GFP nucleus marker32) were cultured in DMEM supplemented with 10% (v/v) fetal bovine calf serum (HyClone), 1% (v/v) GlutaMAX and 1x P/S. Optimization of RNA extraction condition for fixed and permeabilized cells: BC3 cells were harvested, centrifuged at 300g for 3 min to remove the cell media, and washed once with PBS and 1% BSA (molecular biology grade, Gemini Bio-Products).
    p65−/− 3T3
    suggested: None
    Software and Algorithms
    SentencesResources
    Alignment was performed using STAR aligner (version 2.7.3a) and counted using featureCounts.
    STAR
    suggested: (STAR, RRID:SCR_015899)
    featureCounts
    suggested: (featureCounts, RRID:SCR_012919)
    Read subsampling and read mapping: In the FD-seq and methanol fixation comparison experiment, we used samtools view command34 to subsample the output BAM file from Drop-seq tools version 2.3 DetectBeadSynthesisErrors command.
    samtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    To calculate the proportion of reads mapped to different genomic regions, we used Picard CollectRnaSeqMetrics command on the output BAM file directly from the STAR aligner.
    Picard
    suggested: (Picard, RRID:SCR_006525)
    To find the enriched KEGG pathways, we uploaded the list of genes that were upregulated in TMEM119 overexpression versus GFP overexpression (log2 fold change > 1, FDR < 0.05) to g:Profiler26.
    KEGG
    suggested: (KEGG, RRID:SCR_012773)
    RNA velocity analysis: The output files of Drop-seq tools DetectBeadSynthesisErrors function were processed with the dropEst pipeline40 to tag spliced and unspliced transcripts, and the results were analyzed with Python velocyto package33.
    dropEst
    suggested: None
    Python
    suggested: (IPython, RRID:SCR_001658)
    Data availability: The raw data, metadata and count data are deposited in NBCI’s Gene Expression Omnibus (
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

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  4. Version published to 10.1101/2020.09.17.302232v1 on bioRxiv