title: GSM4677544: T-M6T2 T-M7Pool T-M7T1 T-M7T2 T-M7T3 T-M7T4 M-M13N1 M-M12N1 M-M12N2 M-M13N1 M-M12N1 M-M12N2; Mus musculus; ATAC-seq
library_construction_protocol: Lungs were dissociated with fine scissors and then proteolytic digestion was performed using the Lung Dissociation kit (Miltenyi Biotech) following the manufacturer's instructions. Dissociated cells were then incubated at 37 degrees C for 20 minutes with rotation, then filtered using a 100-μm strainer. Red blood cells were lysed using ACK buffer (Thermo Scientific) and stained with 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. Cells were then processed for sciATAC-Seq profiling following 0.1% fixation. The PCR reaction was carried out at the following conditions: 72 °C for 5 minutes (extension), 98 °C for 5 min, and then thermocycling at 98 °C for 10 s, 70 °C for 30 s and 72 °C for 1 min. After thermocycling for 5 cycles, we took 5 µl sample from a few randomly selected wells and added 10 µl of PCR cocktail with 0.6x SYBRgreen. The 15 µl reactions were amplified to saturation to determine the number of cycles required for the remaining samples on the plate. Libraries were amplified for 13-14 cycles in total. Libraries were pooled and purified using Qiagen MinElute PCR purification column. The libraries were quantified using KAPA library quantification kit. Libraries were sequenced on the Next-seq platform (Illumina) using a 150-cycle kit (Read 1: 47 cycles, Index 1: 36 cycles, Index 2: 36 cycles, Read 2: 47 cycles).
attributes.tag: GEO Accession
attributes.value: GSM4677544