ATAC-Seq of U2OS human osteosarcoma cells infected with mycoplasma
100,000 U2OS cells infected with Mycoplasmawere lifted with Tryple-E and pelleted at 400g for 5 minutes. Cells were lysed using a dounce homogenizer (Sigma, D8938) in 1 ml lysis buffer (20mM Tricine-KOH, 25mM MgCl2, 0.25 M sucrose, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, 0.3 % IGEPAL-630, EDTA-free protease inhibitor) for 10 strokes. The homogenate was filtered through a 40um strainer into a 50ml falcon tube, transferred to a 1.5ml eppendorf tube, underlaid with a cushion buffer (0.5 mM MgCl2, 0.5 mM DTT, EDTA-free protease inhibitor, 0.88 M sucrose), and centrifuged at 3000g for 20 minutes at 4C. The pellet was washed in 500l cold PBS and re-pelleted at 700g for 10 minutes. The pellet was resuspended in transposition reaction mix (25 L 2x TD Buffer (Illumina Cat #FC-121-1030); 2.5 L Tn5 Transposase (Illumina Cat #FC-121-1030); 22.5 L Nuclease Free H2O) and incubated for 30 minutes at 37C for stransposition to occur. DNA was then purified using the Qiagen MinElute Kit and eluted with 23 L warm 10 mM Tris pH8. The fragments were amplified, and Illumina adapters added by preparing the following mix (10 L Transposed DNA; 10 L Nuclease Free H2O; 2.5 L 25 M Customized Nextera PCR Primer 1 (Barcodes given in supplementary data); 2.5 L 25 M Customized Nextera PCR Primer 2 (Sigma Barcode Ad1_noMX); 25 L Fast Sybr Green 2x PCR Master Mix (Qiagen Cat #4385612)) and subjected to 15-17 cycles of steps 3-5 ((1) 72C, 5 min; (2) 98C, 30 sec (3) 98C, 10 sec; (4) 63C, 30 sec; (5) 72C, 1 min; Libraries quantified using the KAPA library quantification kit (KK4824), pooled and diluted to 20pM, and sequenced on the Illumina NextSeq 500 system with a 70 cycle high output cartridge.