How do I recalculate "median reads/UMI" after filtering out beads not covered by tissue?

In certain cases, your tissue may not cover the whole tile and there will therefore be beads with background levels of signal that will affect and lower the value of "median reads/UMI".

1. To recalculate this, you will need to first filter out the beads not covered by tissue

2. Then to recalculate the "median reads/UMI":

median reads/UMI = a/b

where

a = median number of reads per bead

b = median number of UMI per bead

The "per bead" values of the "number of reads" and "number of UMI" are located in the seurat object as metadata as $numReads and $nCount_RNA.

Once you have determined the new "median reads/UMI" value, you can determine how saturated your sample was and calculate how much additional sequencing will be needed. We still recommend a value of 8-10 for this metric.