The following 2 days, cells were cultured in S1-media supplemented with final 100 ng/ml Activin-A. After 24 h, culture medium was replaced with S1-media (Final 11,6 g/L MCDB131, Sigma Aldrich, M8537-1L 2 mM D-+-Glucose, Sigma Aldrich, G7528-250G 2,46 g/L NaHCO3, Sigma Aldrich, S5761-500G 2% FAF-BSA, Proliant Biologicals, 68700-1 1:50000 of 100 x ITS-X, Thermo Fisher Scientific, 51500056 1 x GlutaMAX, Thermo Fisher Scientific, 35050-038 0,25 mM ViatminC, Sigma-Aldrich, A4544-100G 1% Pen-Strep, Thermo Fisher Scientific, 15140122) supplemented with final 100 ng/ml Activin-A (R&D Systems, 338-AC-01M) and 1,4 µg/ml CHIR99021 (Stemgent, 04-0004-10). hiPS cell culture) were seeded at a density of 5 x 105 cells /cm2 in mTeSR1 supplemented with 10 µM Y-27632. Briefly, single cell suspensions of ZIP13K2 hiPSCs (s. Pancreatic progenitor (PP) differentiation was performed as previously described 4 with minor changes. Single cell suspensions of definitive endoderm (EN) differentiated cells were utilized for further downstream analysis (qPCR, Western Blot, FACS etc.). Cells were then collected at required timepoints by washing the plate with DPBS before single-cell dissociation was performed with Accutase for 15 min at 37☌, 5% CO2. Media change using the STEMdiff Trilineage Endoderm Differentiation media was performed on a daily bases according to the manufacturer’s instructions. Single cell suspensions of mTeSR1 maintained ZIP13K2 hiPSCs were seeded into the respective culture formats according to the required cell-number as recommended by the manufacturer’s instructions. To guarantee high reproducibility, constant media-quality, and mTeSR1 compatibility, definitive endoderm differentiations were exclusively performed utilizing the STEMdiff Trilineage Endoderm Differentiation media (Stemcell Technologies, 05230).

Wash-steps were performed by spinning cell-suspensions at 300 x g 5 min at room temperature (RT). Cell counting was performed using a 1:1 diluted single-cell suspensions in 0,4% Trypan Blue staining-solution (Thermo Fisher Scientific, 15250061) on the Countess II automated cell-counter (Thermo Fisher Scientific). Single cell splitting was performed by incubating the cells with Accutase (Sigma-Aldrich, A6964) supplemented with 10 µM Y-27632 (Tocris, 1254) for 15 min at 37☌, 5% CO2. Clump-based cell splitting was performed by incubating the cells in final 5 mM EDTA pH 8,0 (Thermo Fisher Scientific, 15575-038) in DPBS (Thermo Fisher Scientific, 14190250) 5 min at 37☌, 5% CO2. ZIP13K2 hiPSCs were maintained in mTeSR1 (Stemcell Technologies, 85850) on pre-coated culture ware (1:100 diluted Matrigel (Corning, 354234) in KnockOut DMEM (Thermo Fisher Scientific, 10829-018)). Sample 44_ChIPseq SOX17 IP day5 sgLNCSOX17 rep1Ĭhromatin Immunoprecipitation seq for SOX17 of day5 CRISPRi cells expressing sgLNCSOX17 gRNA GEO help: Mouse over screen elements for information.

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