title: GSM3604371: SBR OCT4 ChIP; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were pelleted and washed with 1X ice cold PBS at 800g for 5 min. Cells were gently resuspended in 50 μl of ice-cold ATAC lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40), and immediately pelleted at 800g for 10 min at 4°C. To transpose open chromatin regions, cells were resuspended in 50 μl of transposition reaction mix containing 0.5 μM of Tn5 transposase (in-house prepared) in TAPS-DMF buffer (10 mM TAPS-NaOH, 5 mM Mgcl2, 10% DMF) and incubated at 37°C for 30 min. The transposed DNA was purified using a DNA purification kit and eluted in 12 μl of water. A 65 μl PCR reaction was setup with 10 μl of transposed DNA, 0.5 μM of forward primer Ad1_noMX, 0.5 μM of multiplexing reverse primer Ad2.x (Buenrostro et al. 2013), 0.6x SYBR Green I, and 1x PCR Master Mix. The samples were thermocycled at 72°C for 5 minutes, 98°C for 30 s, followed by 12 cycles of 98°C for 10 s, 63°C for 30 s and 72°C for 1 min. The amplified ATAC libraries were purified using a DNA purification kit and size selected using Agencourt AMPure beads (0.55X unbound fraction followed by 1.2X bound fraction).
attributes.tag: GEO Accession
attributes.value: GSM3604371