title: GSM6925069: ATAC-seq_D7_Non-IROrganoids_TGFB1_rep2; Mus musculus; ATAC-seq
library_construction_protocol: Primary cultured organoids were collected by removing Matrigel using cold PBS. Matrigel droplets containing organoids were disrupted. Organoids were pelleted by centrifugation at 300 g for 3 minutes at 4 °C and washed with cold PBS. After removing the PBS and Matrigel, organoids were resuspended in 1 mL pre-warmed (37 °C) TrypLE, and rotated at 37 °C for 15 to 30 minutes until organoids were dissociated into single cells (confirmed via microscope). Cells were pelleted at 300 g for 3 minutes at 4 °C, and washed with cold PBS. Cells were passed through SP Bel-Art Flowmi 40 μm cell strainer and collected into a new protein LoBind tube. 30,000 cells were prepared as mentioned above and used for ATAC-seq as described previously (Buenrostro et al., 2013; Buenrostro et al., 2015) with slight modifications. Briefly, cells were centrifuged at 500 g for 5 minutes at 4°C and resuspended in ice-cold lysis buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.1% NP-40). Cells were then centrifuged at 500 g for 10 minutes at 4°C. The isolated nuclear pellets were incubated with a 50 μl reaction of Tn5 Transposase (Illumina Tagment DNA Enzyme and Buffer Large Kit 20034198) for 30 minutes at 37°C. The transposed chromatin was purified with MinElute PCR purification kit (QIAGEN REF 28004), and PCR was amplified with high-fidelity 2X PCR Master Mix (New England Biolabs M0541). One-third of the maximum fluorescent intensity during a qPCR trial run was used to determine the additional cycles for library prep. NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs).