Viability assays
The viability of PCIS were assessed
using lactate dehydrogenase (LDH)
release (cytotoxicity) assays and water-soluble tetrazolium salt (WST-1)
(metabolic) assays (Roche, Mannheim, Germany) from 0-24h in culture.
Treatment with the detergent
Triton X-100 (MilliporeSigma, Darmstadt, Germany) was included to show
maximum LDH release and as a dead-control.
Single cell
nuclear sequencing of human colon tissue
Colon tissue samples (n=4), also used to generate PCIS, were snap-frozen
and stored at -80oC prior to single nuclear RNA
sequencing (scNucSeq). After library generation and quality control, the
samples were deep sequenced using a NovaSeq (target 10 000 nuclei, 80
000 reads/nuclei). FASTQ files were aligned to a reference human
transcriptome using 10X Genomics Cell Ranger (CA, USA). The resulting
gene expression data were merged using the Seurat R
package.23,24 Batch effects were removed by
“regressing out” variables, and clustering was performed using the
Leiden algorithm implemented in Seurat. Cell identities were assigned
and confirmed using a list of pre-defined marker genes in reference to
single cell gene expression databases and the top distinct genes
expressed by each cluster.25,26