GSM9532919: 26757X3 1311G KNH R1; Mus musculus; ATAC-seq
ATAC-seq was performed on organoids using an Omni-ATAC-based protocol adapted from published methods. Organoids were dissociated into single cells using established protocol, depleted of dead cells by two sequential rounds of magnetic separation using the Dead cell removal kit from (Miltenyl Biotec), and washed in PBS. Cell viability exceeded 90% for all samples. Cells were manually counted, and  100,000 cells per condition were pelleted at 500g for 5 minutes at 4°C in PBS supplemented with 1% BSA. For nuclei isolation, cell pellets were resuspended in cold lysis buffer and incubated on ice for 3 minutes. After addition of wash buffer, nuclei were pelleted at 500g for 10 minutes at 4°C and gently resuspended in transposition mix lacking Tn5. Nuclei were counted, and 50,000 nuclei per reaction were used for transposition. Transposition was performed with 2.5uL of Illumina Tn5 and incubated at 37°C for 30 minutes with shaking at 1,000 rpm. Transposed DNA was purified using Zymo DNA Clean and Concentrator and eluted in 21 µL elution buffer. Libraries were amplified using Q5 Hot Start High-Fidelity Master Mix (NEB) and Nextera barcoded primers. Initial amplification was performed for five cycles, followed by SYBR Green–based qPCR on 10% of each library to determine the minimum number of additional cycles required. Final libraries were purified using Zymo DNA Clean and Concentrator. Library concentration was quantified by Qubit, and size distribution was assessed by Tape Station. Libraries were sequenced using paired-end 150 bp reads on an Illumina platform.