Single cell RNA sequencing of human tissue along the stomach-intestinal tract

Enteroendocrine cells (EECs) are gut epithelial cells that respond to intestinal contents by secreting hormones, including incretins GLP-1 and GIP, which regulate multiple physiological processes. Hormone release is controlled through metabolite-sensing proteins. Low expression, interspecies differences, and existence of multiple EEC subtypes have posed challenges to the study of these sensors. We describe differentiation of stomach EECs to complement existing intestinal organoid protocols. CD200 emerged as a pan-EEC surface marker, allowing deep transcriptomic profiling from primary human tissue along the stomach-intestinal tract. We generated loss-of-function mutations in 22 receptors and subjected organoids to ligand-induced secretion experiments. We delineate the role of individual human EEC sensors in hormone secretion, including GLP-1. These represent potential pharmacological targets to influence appetite, bowel movement, insulin sensitivity and mucosal immunity.

Methods
Human stomach and intestine library preparation, and sequencing

Using BD FACS Fusion single cells were sorted into 384-well hardshell plates containing CELseq2/SORT-seq1, primers. Post sorting, plates were sealed (Greiner, SILVERseal sealer, 676090) and spun down at 2,000 revolution centrifugal force (r.c.f.) for 2 minutes (Eppendorf 5810R). Plates were stored at −80 °C before being processed. All plates but plate JB-s050 were processed according to the VASA-plate library preparation protocol, previously published Plate JB-s050 was processed using the CEL-Seq2 library preparation protocol. Samples were sequenced on a NextSeq 2000 with the following parameters: 150 000 reads/cell, Read 1 26 cycles (Index 6 cycles), Read 2 60 cycles. Plate AM003 was an exception and for which Read 1 30 cycles, and Read 2 120 cycles. FASTQ file pre-processing (VASA-plate) and mapping of VASA data was performed according to previously published methods. In short, read 1 contains the 6 nt long UMI and cell-specific barcode (one unique barcode per well in a 384-well plate), and is thus used to assign reads to a specific cell/well. Read 2 was trimmed and mapped to the human GRCh38 genome (Ensembl 99). Data from all plates were mapped to the genome indexed for 60 nucleotides.

Libraries were generated following the protocol described for VASA-Seq  https://doi-org.utrechtuniversity.idm.oclc.org/10.1038/s41587-022-01361-8

For references see original manuscript https://www.science.org/doi/10.1126/science.adl1460