CHD3 and CHD4 coordinate gene expression programs to maintain ß-cell function and identity in vivo
To establish the role of CHD3 and CHD4 on controlling human gene expression programs in the islet, we generated pseudoislets from non-diabetic human donors that were treated with shRNA for CHD3, CHD4 or CHD3+CHD4. mRNA-Seq was performed on isolatd RNA from pseudoislets Overall design: Human pseudoislets were generated from 12,000 IEQ donor islets using a modified reaggregation protocol. Upon arrival, islets were resuspended and cultured overnight in Standard CMRL 1066 medium (Corning, 15-110-CV) under standard conditions. The following day, floating islets were collected, washed twice with 2mM EDTA in Ca²?/Mg²?-free PBS, pelleted at 200g and enzymatically dissociated into a single-cell suspension using 0.025% trypsin for 8–9 min with gentle pipetting. The reaction was quenched with an equal volume of VPM, and cells were pelleted, washed once in VPM, and resuspended by gentle flicking to minimize shear stress. For knockdown, dispersed cells were transduced with lentivirus encoding shControl, shCHD3, shCHD4, or shCHD3+shCHD4 at a multiplicity of infection (MOI) of 15 by co-incubation in non–tissue culture-treated 4-well plates (Nunc, 179820) for 3 h at 37°C. Cells were then washed twice with VPM and seeded into ultra-low attachment round-bottom 96-well plates (Nunclon Sphera, PerkinElmer 174925) at 2000 cells per well to allow reaggregation. After 7 days, RNA from pseudoislets were harvested for mRNASequencing