catcheR_10Xnocatch - Identify cells expressing no shRNA

This optional step identifies and annotates cells transduced with a cassette lacking the shRNA. These “empty” cells can be included as negative controls in downstream analyses.

In the same working directory used for catcheR_10Xcatch(), run:

catcheR_10Xnocatch(
    group = c("docker", "sudo"),
    folder,
    expression.matrix,
    threshold,
    sample = 1,
    reference = "TACGCGTTCATCTGGGGGAGCCG"
)

Arguments:

  • group: either “docker” or “sudo”, depending on user permissions (see: https://docs.docker.com/engine/install/linux-postinstall/)

  • folder: path to the working directory

  • expression.matrix: filename of the cell-by-gene count matrix (CSV format)

  • threshold: integer number of UMIs supporting the “empty” reference required to classify a cell as empty

  • sample: integer sample index (default = 1)

  • reference: string of the reverse complement of the empty vector’s unique identifier (default is the 3’ end of the OPTtetR cDNA)

Example usage:

catcheR_10Xnocatch(
    group = "docker",
    folder = "path/to/folder",
    expression.matrix = "filename.csv",
    threshold = 5
)

Output:

  • An updated silencing_matrix.csv (also in RDS format), where empty cells are annotated using the following format:

cellID_?_empty_NA_empty

This enables easy tracking of negative control cells for further analysis.