GSM4190695: K562_vector_CPT1000_4h [K562Vec-1000]; Homo sapiens; ATAC-seq
Fifty thousand cells were treated with DMSO or camptothecin for the indicated time. To prepare nuclei, cells were lysed using cold lysis buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630). Immediately following the nuclei preparation, the pellet was re-suspended in the transposase reaction mix [25 μl 2x TD buffer, 2.5 μl Transposase (Illumina) and 22.5 μl of nuclease free water], and incubated for 30 minutes at 37 °C. Directly following transposition, the sample was purified using a DNA Clean and Concentrator-5 (ZYMO RESEARCH) and eluted with 25 μl of DNA elution buffer. Following purification, we amplified library fragments using 1x NEBnext PCR master mix and 1.25 μM of custom Nextera PCR primers 1 and 2, using the following PCR conditions: 72°C for 5 minutes, 98°C for 30 seconds, followed by thermo-cycling at 98°C for 10 seconds, 63°C for 30 seconds and 72°C for 1 minute. We amplified the full libraries for 5 cycles, after 5 cycles we took an aliquot of the PCR reaction and added 10 μl of the PCR cocktail with Syber Green at a final concentration of 0.6x. We ran this reaction for 20 cycles, to determine the additional number of cycles needed for the remaining 45 μL reaction. Libraries were amplified for a total of 10–12 cycles and purified using a PCRClean DX yielding a final library concentration of ~30 nM in 20 μl. Illumina Genomic DNA protocol (Illumina Paired-END DNA Sample Prep Kit).
GEO Accession
GSM4190695