Bulk and single-cell RNA-seq of human fetal pancreatic organoids

The mammalian pancreas consists of three epithelial compartments: the acini and ducts of the exocrine pancreas and the endocrine islets of Langerhans. Murine studies indicate that these three compartments derive from a transient, common pancreatic progenitor. Here, we report the derivation of 18 human fetal pancreas organoid (hfPO) lines from gestational week 8-17 (8-17GW) fetal pancreas samples. Four of these lines, derived from 15-16GW samples, generate acinar-, ductal- and endocrine lineage cells while expanding exponentially for >2 years under optimized culture conditions. Single-cell RNA sequencing identifies rare LGR5+ cells in the fetal pancreas and hfPOs as the root of the developmental hierarchy. These LGR5+ cells share multiple markers with adult gastrointestinal tract stem cells. Organoids derived from single LGR5+ organoid-derived cells recapitulate this tri-potency in vitro. We describe a human fetal tri-potent stem/progenitor cell, capable of long-term expansion in vitr..., Bulk RNA sequencing Human fetal pancreatic organoids were cultured in expansion, endocrine, or acinar cell differentiation conditions for 10 days. 
Organoids were collected and RNA extraction was performed according to the manufacturer's instructions. RNA integrity was determined using the Agilent RNA 6000 Nano kit with the Agilent 2100 Bioanalyzer (Agilent). RNA integrity (RIN) values ranged from 9.0–10.0. Samples used for bulk RNA sequencing did not have a RIN <9.0. RNA concentrations were determined using the Qubit RNA HS Assay Kit (Thermo Fisher). RNA libraries were prepared with the TruSeq Stranded messenger RNA polyA kit and paired-end (2 × 50 base pairs) sequenced on an Illumina NextSeq 2000. Reads were mapped to the human GRCh38 genome assembly using STAR (DOI: 10.1093/bioinformatics/bts635).
Single-cell RNA sequencingFor scRNA-seq cells were FACS sorted into 384-well plates pre-printed with primers (Single Cell Discoveries) and stored at -80°C until used for library preparat..., , # Bulk and single-cell RNA-seq of human fetal pancreatic organoids

# **Bulk RNA sequencing**

Human fetal pancreatic organoids (hfPOs) were cultured in expansion, endocrine, or acinar cell differentiation conditions for 10 days. 

Organoids were collected and RNA extraction was performed according to the manufacturer's instructions. RNA integrity was determined using the Agilent RNA 6000 Nano kit with the Agilent 2100 Bioanalyzer (Agilent). RNA integrity (RIN) values ranged from 9.0–10.0. Samples used for bulk RNA sequencing did not have a RIN <9.0. RNA concentrations were determined using the Qubit RNA HS Assay Kit (Thermo Fisher). RNA libraries were prepared with the TruSeq Stranded messenger RNA polyA kit and paired-end (2 × 50 base pairs) sequenced on an Illumina NextSeq 2000. Reads were mapped to the human GRCh38 genome assembly using STAR (DOI: [10.1093/bioinformatics/bts635](https://doi.org/10.1093/bioinformatics/bts635)).

## Datasets

File: Bulk_OrgsCystVSbud\
Bulk-seq of h...