GSM7978636: Day7_vAFG_B1; Homo sapiens; RNA-Seq
Day-3 meso-endoderm spheroids and day-7 vascularized AFG and MHG organoids were dissociated with TrypLE Express Enzyme (1X) (Invitrogen) at 37oC for 7 – 12 mins. The reactions were stopped by adding N2B27 basal medium and centrifuged and filtered with 40um strainer. Day-21 vascularized gut tube organoids were embedded in a Collagen-Matrigel mixture, so were dissociated with a different digestion cocktail. Briefly, the organoids from one trans-well insert were immersed in 1ml Cell Recovery Solution at 4oC for 5 mins and triturated with P1000 wide-bore tip to break the matrix. The solution was centrifuged and resuspended in 1ml pre-warmed Dispase (5mg/ml)/Liberase solution (0.5mg/ml), and the organoids were minced with scissors. The mixture was incubated at 37oC for 20 mins with gentle triturate every 5 – 7 mins. After the first 5 mins, 10ul Dnase I (60U/ml) was added to the reaction. To stop the reaction, 9ml of Knock-out DMEM/F12 containing 10% FBS was added and centrifuged and filtered with 40um strainer. Cell pellets were resuspended in N2B27 basal medium and loaded to 10X Chromium controller for 3' single-cell RNA-seq (v3.1). Library was performed according to the manufacturer's instructions (Single cell 3' v3.1, 10x Genomics).