GSM8989494: ATAC-seq K562, empty vector control, replicate 2; Homo sapiens; ATAC-seq
Extraction followed the standard Active Motif protocol for ATAC-seq of cryopreserved samples; Supplier: Active motif, Catalogue number: 53150; Cell sample Preparation 1. Count cells and aliquot 50,000 to 100,000 cells into a fresh 1.5 mL centrifuge tube for each sample. 2. Centrifuge the cells at 500 x g for 5 minute at 4°C. Orient the tubes such that the pellet will be where you expect it to be. If there is no visible pellet, spin an additional 5 minutes at 1,000 x g at 4°C. 3. Gently remove supernatant by pipetting and add 100 μL of ice-cold PBS. Do not resuspend or disturb pellet. Spin once more at 500 x g for 5 minutes at 4°C. 4. Remove supernatant very carefully, ensuring to not disturb the cell pellet. By pipetting, thoroughly resuspend the cell pellet in 100 μL ice-cold ATAC Lysis Buffer. 5. Transfer the resuspended cell pellet to a PCR tube on ice. Immediately spin down at 500 x g for 10 minutes at 4°C. During this time, prepare the Tagmentation Master Mix. 6. After the spin, remove the supernatant very carefully, ensuring the cell pellet is not disturbed (this is a critical step; take extra care to remove the ATAC Lysis Buffer but not lose nuclei). Immediately continue to the Tagmentation Reaction and Purification steps. Tagmentation Reaction and Purification 1. Add 50 μL of Tagmentation Master Mix to each sample (does not have to be on ice). Gently pipette to resuspend nuclei in the Tagmentation Master Mix with a multi-channel pipettor. 2. Incubate the tagmentation reaction at 37°C for 30 minutes in a thermomixer (or similar device) set at 800 rpm. 3. Immediately following the tagmentation reaction, transfer each sample to a clean 1.5 mL microcentrifuge tube. 4. Add 250 μL DNA Purification Binding Buffer and 5 μL 3 M sodium acetate to each sample. 5. If the color of the sample is anything other than bright yellow, add additional 3 M sodium acetate in 5 μL increments until the proper color is achieved. 6. Mix each sample by pipetting. 7. For each sample, place a labeled DNA purification column into a collection tube. 8. Transfer each sample to its corresponding column, close the cap, and centrifuge at 17,000 x g (14,000 rpm) for 1 minute. 9. Discard the flow-through and return the collection tube to the column. 10. Add 750 μL of Wash Buffer (see note above in step 9 the Wash Buffer should have a final concentration of 80% ethanol) to the column and cap the column. 11. Centrifuge at 17,000 x g for 1 minute. 12. Discard the flow-through and return the column to the collection tube. 13. With the column cap open, centrifuge at 17,000 x g for 2 minutes to remove residual Wash Buffer from the column. 14. Transfer each column to a new microcentrifuge tube. 15. Add 35 μL of DNA Purification Elution Buffer to the center of the column matrix, cap the column, and incubate for 1 minute at room temperature. 16. Centrifuge at 17,000 x g for 1 minute. 17. Discard column. DNA purification is complete. 18. Purified DNA can be stored at -20°C, or you may proceed directly with the PCR Amplification of Tagmented DNA steps that follow. Library preparation followed the standard Active Motif protocol for ATAC-seq; Supplier: Active motif, Catalogue number: 53150 PCR Amplification of Tagmented DNA  1. Set up the PCR reactions by adding the components in the order shown below. If libraries are to be multiplexed for sequencing on the same flow cell, ensure that a unique i5 and/or i7 index combination is used for each. Each sample will require a combination of one i7 Indexed Primer and one i5 Indexed Primer in the PCR amplification reaction. There are 4 x 4 = 16 unique combinations of i7/i5 primers for a total of 16 samples that can be multiplexed. These Indexed Primers are based on Illumina's Nextera adapters. 2. Perform PCR using the following program on a thermal cycler (with a heated lid): 72°C 5 minutes 98°C for 30 seconds 10 cycles of: 98°C for 10 seconds, 63°C for 30 seconds, 72°C for 1 minute Hold at 10°C. 3. Perform SPRI clean-up with 60 μL SPRI bead solution (1.2X the sample volume), eluting in 20 μL DNA Purification Elution Buffer. Have 400 μL of freshly-prepared 80% ethanol ready per sample: A. Add 60 μL well-mixed, room temperature SPRI Beads to each sample. B. Vortex briefly to mix and incubate for 5 minutes at room temperature to allow beads to bind. C. Apply magnet to collect beads. D. Once the solution is clear, aspirate the supernatant. E. With the magnet still applied to the sample, add 180 μL 80% ethanol to each sample without mixing. F. Incubate for 30 seconds at room temperature. G. Aspirate the supernatant. H. Repeat steps E through G for a second ethanol wash. I. Allow tubes to sit at room temperature so that residual ethanol can evaporate. Once the beads transition from shiny to matte (2-5 minutes), proceed to the next step. J. With the tubes separated from the magnet, add 20 μL DNA Purification Elution Buffer. K. Cap tubes and vortex to mix. L. Incubate samples for 5 minutes at room temperature. M. Apply magnet to collect beads. N. Once the solution is clear, transfer each supernatant containing the eluted DNA to a fresh tube. 4. At this stage, libraries are ready for quantification and sequencing. Use a library quantification kit for next-generation sequencing to quantify the library (e.g. Kapa Biosystems, Catalog No. KR0405). PCR amplified libraries can also be analyzed to assess size distribution with a Bioanalyzer, TapeStation, or similar instrument to assess size distribution.