GSM2945824
GSM2945824: ATAC_seq_JNKi DE Day1-b; Homo sapiens; ATAC-seq
Nuclear Isolation: 50,000 cells were collected in an eppendorf tube, washed with 1mL of cold PBS and spun down at 1500rpm for 5 minutes at 4 oC. Cell were pelleted, and washed with 1mL of ice-cold ATAC Buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2). Then cells were pelleted again and resuspended in 50μL of ATAC Lysis Buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40 or IGEPAL-Ca630) for 2 minutes on ice. 1 mL of cold ATAC Buffer was added to dilute out the lysis buffer. Cells were pelleted by centrifugation with 1500 RPM for 10 minutes at 4oC. Tagmentation: Supernatant was aspirated to leave behind ~22.5 μL sample liquid.  The resuspended pellet was transferred to a PCR tube containing 2.5 μL Tagmentation Enzyme (transposase) and 25 μL of Tagmentation Buffer (Illumina Nextera DNA Sample Preparation Kit). After incubating the reaction for 30 minutes at 37oC, 0.2% SDS was added to the reaction. After incubating the reaction for 5 minutes at room temperature, samples were purified with 2X Agencourt AMPure XP beads (Beckman Coulter A63881). 50μL of sample was mixed with 55 μL of NEBNext Q5 Hot Start HiFi PCR Master Mix (NEB, catalogue M0543L) and 5 μL of primer mix. For the primer mix, we used the Nextera primers The universal primer Ad1 and the unique index primer 2.X  (ex. 2.1, 2.2, 2.3, etc.) were mixed so that each had a final concentration of 25 μM. The samples were amplified with the following PCR program: 65 ºC, 5 minutes; 98 ºC, 30 seconds; 98 ºC, 10 seconds, 65 ºC 30 seconds (12 times). The samples were purified with 1.5X AMPure XP beads and the concentration was checked using PicoGreen and median fragment size was checked using the Agilent D1000 screentape on the Agilent Technologies 2200 TapeStation. Samples were sequenced PE50 on a HiSeq 2500.
GEO
GSM2945824
GEO Accession
GSM2945824