GSM6509093: NeuroD1ST_H3K4me3; Mus musculus; OTHER
The pancreata microdissected from E15.5 embryos were directly trypsinized and prepared for FACS. Briefly, tissue was dissociated using 0.05% trypsin/0.53 mM EDTA at 37°C for 5 min. Digestion was stopped by adding the FACS buffer (2% FBS and 10 mM EGTA in PBS), and cells were then 1x washed by FACS buffer. Finally, cell suspensions were filtered through 40 μm nylon mesh and immediately tdTomato+ cells were sorted using a flow cytometer (BD FACSAriaTM Fusion), through a 100 μm nozzle in 20 psi, operated with BD FACSDiva™ Software. Freshly FACS-sorted tdTomato+ pancreatic endocrine cells were captured by Concanavalin A-coated magnetic beads to facilitate subsequent washing steps. Spike-in (SNAP-CUTANA™ K-MetStat Panel, EpiCypher, 19-1002) was added to the immobilized nuclei in dilution 1:250.  CUT&Tag validated primary antibodies anti-H3K4me3, anti-H3K27me3, normal rabbit IgG negative control, and anti-rabbit secondary antibody were used. Binding of pAG-Tn5 (EpiCypher,15-1017, 2.5μL/reaction) was at RT for 60 min followed by tagmentation. To stop tagmentation and solubilize DNA fragments, 1.67 μL 0.5M EDTA, 0.5 μL 10% SDS and 0.42 μL 20 mg/mL Proteinase K (20mg/mL) was added to each sample. Samples were incubated for 1 hr at 55°C to digest (and reverse cross-links), followed by DNA precipitation and purification. DNA was dissolved in 22 μL of 1 mM Tris-HCl pH 8, 0.1 mM EDTA buffer and utilized as template for library generation with Universal i5 Primer and Uniquely Barcoded i7 Primers for Illumina. 21 μL DNA + 2 μL of 10 μM Universal or barcoded i5 primer + 2 μL of 10 μM uniquely barcoded i7 primer (using a different barcode for each sample) and 25 μL non-hot start CUTANA® High Fidelity 2x PCR Master Mix (EpiCypher, 15-1017) was added to each thin-wall 0.2 ml PCR tube. Library was amplified under CUT&Tag-specific PCR cycling parameters: a)5 min at 58°C; b) 5 min at 72°C; c) 45 sec at 98°C; d) 15 sec at 98°C; e) 10 sec at 60°C; f) 1 min 72°C. Steps "d-e" were repeated 25-27 times. DNA cleanup was performed using 1.3x AMPure beads (Beckman Coulter, A63880). Each purified DNA library for Illumina sequencing was tested on Agilent High Sensitivity DNA Chip. MiSeqReagent Kit v3 (150-cycle) was used for sequencing.