GSM9092552: endocine pancreas,control60_ISL1_E14.5,1; Mus musculus; OTHER
The pancreases from P9 pups were first perfused by 1mg/ml collagenase in Hank's balanced salt solution, dissected, and incubated at 37oC for 10-12 min to release endocrine cells/islets. Digested pancreatic tissue was washed 3x in Hank's solution. To generate single cells, the tissue was further dissociated using trypsinization, as previously described 100. Briefly, tissue was dissociated using 0.05% trypsin/0.53 mM EDTA at 37oC for 5 min. Digestion was stop by the FACS buffer (2% FBS and 10 mM EGTA in PBS), 100100999897and 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. For scRNA-seq, sorted cells were collected into precooled FACS buffer (4°C). The total time from euthanasia to cell collection was approximately 3 hours. For the epigenetic study 〜 20000 cells were sorted. The benchtop CUT&Tag version 3 was performed 38,39 with modifications, as previously published 17,89. Nuclei isolated from freshly FACS-sorted tdTomato+ pancreatic endocrine cells were captured by Concanavalin A-coated magnetic beads to facilitate subsequent washing steps and the reaction was carried out in 0.2 ml PCR tubes. Details of the antibodies used are provided in Supplementary Table 1. Binding of pAG-Tn5 (EpiCypher, 15-1017) 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 at 55 ºC for 1 hr 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. DNA libraries were sequenced on the MiSeq, Illumina using MiSeq Reagent Kit v3, which allows extend read lengths up to 2 x 75bp at the OMICS Genomics facility (BIOCEV).