GSM5461507: NZO-F; Mus musculus; ATAC-seq
After incubating, the islets were lysed by trituration with a 25 gauge needle until intact islets were no longer visible, usually 6 triturations. The lysate was centrifuged at 500 x g for 10 minutes at 4C. This generated a crude nuclei pellet and a supernatant. The supernatant was discarded and the nuclei pellet was washed with 100 uL of ATAC Lysis buffer in order to reduce cytoplasmic and mitochondrial contamination. This mixture was centrifuged at 500 x g for 10 minutes at 4C and the supernatant was removed. Per ATAC-seq sample, a mixture of 25 uL 2x TDE buffer, 22.5 uL nuclease-free water, and 2.5 uL TDE1 transposase enzyme (Nextera DNA Library Prep kit, Illumina) was applied and incubated for 30 minutes in a 37C water bath. The samples were then purified using a MinElute Reaction cleanup kit (Qiagen) and eluted using two sequential aliquots of 10 uL EB buffer.  After purification all ATAC samples were kept at -80C. All ATAC-seq samples were transposed and frozen prior to preparing all libraries. Libraries were amplified using 20 uL of ATAC sample, 2.5 uL Primer-1 (Ad1_noMX, 25 uM working stock), 2.5 uL Primer-2 (Ad2.X, 25 uM working stock), and 25 uL of NEBNext High Fidelity 2x PCR Master Mix. Each ATAC sample was amplified by 12 cycles which was determined by qPCR to be saturating for the libraries. The PCR thermocycler was set to 72C for 5 minutes, 98C for 30 seconds, and then 12 total cycles of 98C for 10 seconds, 63C for 30 seconds, 72C for 1 minute. After amplification the libraries were purified using MinElute PCR purification cleanup kit (Qiagen).