title: GSM8603334: Empty-flag AML12 cell line,Flag, chip; Mus musculus; ChIP-Seq
library_name: GSM8603334
library_construction_protocol: CHIP-seq: for liver tissue, frozen samples were chopped into small pieces, immediately incubated in freshly prepared disuccinimidyl glutarate solution (2 mM in PBS), and rotated for 30 min at room temperature; for cell lines, samples were collected from 10 cm cultured dishes using cell scrapers. Then samples were washed in PBS buffer twice, crosslinked by 1% formaldehyde (in PBS with proteinase-inhibitor cocktail), and fixed on a nutator shaker for 10 min at room temperature. Fixed samples were quenched with 0.125 M glycine and washed with cold PBS buffer with protease inhibitors three times. Ground the liver tissues with a dounce homogenizer on ice, passed the homogenized tissue suspension through a 70 μM cell strainer to remove the connective tissue, and then washed samples with cold PBS buffer with protease inhibitors three times. Samples were then lysed by ice-cold lysis buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate, 1% Triton X-100, 1 mM PMSF and protease inhibitor cocktail) with 0.1% SDS. Lysates were digested using 0.5 μl micrococcal nuclease (MNase) (10011S, CST) in reaction buffer (50 mM Tris, 5mM CaCl2, pH 8.0) for 3 min at 37 °C, and then were stopped with 25 mM EDTA. About 10 μg of the digested chromatin was used for immunoprecipitation. ATAC-seq: Briefly, frozen liver samples were chopped into small pieces, and immediately incubated in homogenization buffer (10mM Tris, 0.25mM sucrose, 25mM KCl, 5mM MgCl2, 0.1 mM DTT, 1% BSA, 0.1% NP 40 with RNase inhibitor). Ground the liver tissue pieces with a dounce homogenizer on ice, passed the homogenized tissue suspension through a 70 μM and 40 μM cell strainer, washed once in cold Nuclei Wash Buffer (20% iodixanol in HB solution), and then resuspended samples in Blocking Buffer (10% BSA in PBS with RNase inhibitor). The segmentation reaction was performed using transposases (Vazyme, TD501) for 30 min at 37 °C. Segmented DNAs were purified and amplified using the TruePrepTM Index Kit (Vazyme, TD202). The ChIP-seq and ATAC-seq libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (New England BioLabs) and NEBNext Multiplex Oligos for Illumina (New England BioLabs). Sequencing was performed on an Illumina NovaSeq 6000.