GSM4308259: ETP_rep1; Mus musculus; ATAC-seq
Mice were euthanized 8 weeks after tumor initiation in an isoflurane chamber. Lungs were inflated by injecting digestion buffer (Adv DMEM/F12, 5uM HEPES, DNase, 1mg/mL Collagenase, 0.36mM CaCl2) into the trachea. Lungs were dissociated with fine scissors and proteolytic digestion was performed using the lung digestion buffer. Dissociated cells were incubated at 37° for 1 hour with rotation. Cells were washed in 1x PBS and red blood cells were lysed using ACK lysis buffer (Thermo Scientific) for 3 minutes at room temperature. Cells were filtered using a 100um strainer and stained with DAPI (Sigma-Aldrich) and APC-conjugated CD45 (BD, 559864), CD11b (eBioscience 17-0112-82), CD31 (Biolegend, 102510), Ter119 (BD, 557909), and DAPI (Sigma-Aldrich). Fluorescence-activated cell sorting (FACS) of immuno-stained primary cells was performed using a FACSAria sorter (BD) to isolate tdTomato+; DAPI-; APC- tumor cells for sciATAC-seq. Library was prepped using the SureCell ATAC-Seq Library Prep Kit (10000106678, Bio-Rad). Tagmented cells were loaded onto the ddSEQ Single-Cell Isolator (12004336, Bio-Rad) and libraries were prepared using the SureCell ATAC-Seq Library Prep Kit (17004620, Bio-Rad) and SureCell ATAC-Seq Index Kit (12009360, Bio-Rad). Bead barcoding and indexing were performed utilizing a C1000 Touch thermal cycler with a 96-Deep Well Reaction Module (1851197, Bio-Rad); with a 96-Deep Well Reaction Module (1851197, Bio-Rad). The PCR conditions for indexing were: 37 °C for 30 min, 85 °C for 10 min, 72 °C for 5 min, 98 °C for 30 s, eight cycles of 98 °C for 10 s, 55 °C for 30 s and 72 °C for 60 s, followed by a single 72 °C extension for 5 min. Products were cleaned up using Ampure XP beads (A63880, Beckman Coulter). Further amplication was completed using PCR conditions: 98 °C for 30 s, seven cycles at 98 °C for 10 s, 55 °C for 30 s and 72 °C for 60 s, followed by a single 72 °C extension for 5 min. PCR products were purified using Ampure beads before proceeding to sequencing.