title: GSM9078051: Primary Lung Cancer EML-ALK V3, sgSetd2 Sample5; Mus musculus; ATAC-seq
library_name: GSM9078051
library_construction_protocol: Individuals tumors were microdissected and minced into <0.5 mm3 pieces and transferred into pre-warmed digestion media (Dispase (5 U/mL)), DNase I, Grade II (1 mg/mL), Y-27632 ROCK inhibitor (0.5 mM) in Advanced DMEM/F-12. The minced tumors were digested at 37°C for 40 minutes with gentle horizontal shaking. Tumors were vigorously disrupted by pipetting every 15 minutes.   The   digestion   media   was   then   neutralized   with   stop   media   (DMEM/F-12), FBS (10%), HEPES (0.01 M) with DNase I, Grade II (0.1 mg/mL), and ROCK inhibitor (0.05 mM)) and cell suspensions were passed through 40 μm cell strainers. The samples were centrifuged at 1,100 rpm for 7 minutes and pellets were resuspended in red blood cell lysis buffer (Sigma-Aldrich, R7757). Cells were washed with FACS buffer by spinning at 1,100 rpm for 7 minutes. Cell pellets were then resuspended in an appropriate volume of FACS buffer and sorted for Tdtomato-expressing cells Between 6,000 and 25,000 cells were isolated by FACS and frozen in 500 μL Bambanker freezing media. Cells were thawed, washed once in 1 mL cold PBS, and resuspended in 50 μl cold lysis buffer (Buenrostro et al., 2015). Nuclei were centrifuged for 1 minute at 500 g at 4 °C and transposed with adaptor loadedTn5 transposase for 30 minutes. DNA was purified using a Zymo DNA Clean and Concentrator 5 kit (Zymo D4004). ATAC-Seq libraries were prepared using NEBNext High-Fidelity 2× PCR Master Mix (NEB M0541) as described (Buenrostro et al., 2015). Purified libraries were assessed using Agilent High Sensitivity DNA 1000 Tapestation Kit (Agilent). Paired-end 150 bp reads were sequenced with Novogene on the NovaSeq XPlus platform at approximately 5 x 107 paired-end reads per sample