ATAC-seq of xenograft: HuH7 tumor
Library preparation for ATAC-sequence of nuclei isolated from Snap frozen tumors were conducted with modified reported protocols (OMNI-ATAC). All of steps were carried out at 4C. Briefly, a frozen tissue fragment (20-50 mg) was placed into a pre-chilled 2-mL Dounce homogenizer containing 1-2 mL of Homogenization buffer (HB). Tissue was homogenized with strokes with loose A pestle and connective tissue and residual debris were removed by filtration with 70um nylon mesh filter, followed by strokes with tight B pestle, and filtered with 70um nylon mesh filter again, followed by centrifugation. The pellet was suspended in HB and nuclei were collected with Iodixanol density gradient centrifugation (25% (nuclei suspension and 50%idioxanol sol [1:1]), 29% and 35% iodixanol solution were layered underneath, respectively). After the centrifugation, layer between 29% and 35% solution were collected and count number of nuclei with trypan blue. 50,000-100,000 nuclei were applied to following steps. Library preparation of ATAC-sequence were conducted in duplicate after at the Tagmentation steps from two independent tumor samples. For following steps were conducted with ATAC-Seq kit (ActiveMotif, California, USA) along with manufactures instruction. NGS assay were conducted Pair-end sequencing of 2 x 76 bp was conducted using an illumina NextSeq 550 according to the manufacturers instructions.
SUB14791544
ATAC-seq_4_1