See growth protocol Cells were grown in RPMI 1640 medium lacking L-Glutamine (Corning #15-040-CM), supplemented with 15% fetal bovine serum, 1% GlutaMAX (ThermoFisher #35050061) and 1% penicillin-streptomycin solution (ThermoFisher #10378016) at a density of 0.5x106 to 1.0x106 cells/mL. Cells were passaged every 2-3 days to maintain this density. Cells were harvested for ATAC-seq by centrifugation at 500 x g for 5 minutes at 4°C and resuspended in PBS. Cells were counted using a hemocytometer and 50,000 cells were immediately placed into a 1.5 mL Eppendorf tube for ATAC-seq.  For the “no-detergent” samples, Igepal-CA630 was removed from the lysis buffer and replaced with water. For the anti-mt CRISPR treatment, we estimated that half of the DNA in each library was of mitochondrial origin, thus a 20 nM ATAC-seq library contained a mtDNA target concentration of 10 nM. Based on this value, 40, 100, 200, or 400 molar excess of Cas9 enzyme was used (NEB #M0386M) along with 100 molar excess gRNAs in a 30 μl reaction. The reaction was set up according to the protocol for Cas9 (NEB). Briefly, the appropriate amount of Cas9 enzyme and gRNAs were mixed with 3 μl of 10X Cas9 Buffer and water to a final volume of 22 μl. This was incubated at 25°C for 10 minutes and then 8 μl of the ATAC-seq library was added and the reaction was incubated at 37°C for one hour. For the two-hour treatment, the incubation was extended an additional hour; for the “Cas9 boost” treatment, the same amount of Cas9 enzyme was added after 1 hour of incubation and left for an additional hour. Reactions were subsequently treated with 1 μl of 20mg/mL Proteinase K for 15 minutes and purified using a Qiagen MinElute kit followed by elution in 10 μl nuclease-free water. Treated libraries were run on a high sensitivity Bioanalyzer chip to assess fragment size distribution and concentration. Because the multiplexing barcodes are added before treatment, for each batch of experiments, samples were sequenced on two lanes of an Illumina Hi-Seq 4000 instrument, separating anti-mt-CRISPR untreated and treated samples. ATAC-seq was performed according to Buenrostro et al. Nature Methods 10, 1213-1218 (2013). For the “no-detergent” samples, Igepal-CA630 was removed from the lysis buffer and replaced with water.  NatureFor the “no-detergent” samples, Igepal-CA630 was removed from the lysis buffer and replaced with water.
Experimental Factor: yes
detergent
Experimental Factor: 100
cas9 x
Experimental Factor: 1000
grna x
Experimental Factor: 1
incubation time
Experimental Factor: no
cas9 boost