For RNA isolation from stomach tissues, (i) pediatric stomach biopsies consisted of only mucosal layer from surgical samples; (ii) Late fetal stomachs were cut open and mucosal layer was isolated; and (iii) early fetal stomachs were processed with no layer isolation, given the small size of the samples. Mucus was removed from all the samples with a glass coverslip to prevent RNA loss during the isolation protocol, and tissues washed in ice-cold PBS. Then the tissues were finely cut with a scalpel on a Petri-dish on ice and transferred to 1.5 mL tubes.
RNA was isolated from cultured organoids in Matrigel with 30 min treatment of the droplets with Cell Recovery Solution (Corning) at 4 °C. Cells were then washed in ice-cold PBS to remove matrix leftovers that could interfere with RNA isolation. Organoids were centrifuged at 200×g at 4 °C and supernatant discarded.
Dry pellets of tissues and organoids were lysed with RLT buffer (Qiagen). RNA was isolated with RNeasy Mini Kit (Qiagen) following manufacturer’s instructions. Total RNA was quantified using the Qubit 2.0 fluorimetric Assay (Thermo Fisher Scientific).
RNA was isolated from cultured organoids in Matrigel with 30 min treatment of the droplets with Cell Recovery Solution (Corning) at 4 °C. Cells were then washed in ice-cold PBS to remove matrix leftovers that could interfere with RNA isolation. Organoids were centrifuged at 200×g at 4 °C and supernatant discarded.
Dry pellets of tissues and organoids were lysed with RLT buffer (Qiagen). RNA was isolated with RNeasy Mini Kit (Qiagen) following manufacturer’s instructions. Total RNA was quantified using the Qubit 2.0 fluorimetric Assay (Thermo Fisher Scientific).