GSM6718844: KO3; Mus musculus; ATAC-seq
A total of 27,000-80,000 Tomato+ mature β-cells were collected for ATAC-Sequencing. Cells were first lysed to collect nuclei by incubating with 100 μL lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.10% Tween-20, 0.10% Nonidet P40 Substitute, 0.01% Digitonin, 1% BSA, 1 mM DTT, 40 U/uL RNase Inhibitor) for 3 minutes, centrifuging at 300 rcf for 5 minutes at 4°C, and discarding the supernatant. The resulting nuclei were then washed with 1 mL wash buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.10% Tween-20, 1% BSA, 1 mM DTT, 40 U/μl RNase Inhibitor) and centrifuged at 200 rcf for 5 minutes at 4°C for 2-3 times. The final nuclei, ranging from 14,000-45,000 in number, were used for tagmentation and library preparation Nuclei were immediately suspended in 50 μL tagmentation reaction using 2xTD buffer (Illumina, FC-121-1030) and Nextera Tn5 Transposase enzyme (Illumina, FC-121-1030). Fragments greater than 600 bp were excluded in library preparation. Each resulting indexed library was quantified and its quality assessed by Qubit and Agilent Bioanalyzer, and multiple libraries pooled in equal molarity