GSM6934463: ATAC-Seq PHD-fl/fl Untreated Rep3; Mus musculus; ATAC-seq
ATAC-seq was performed using OMNI-ATAC supplementary protocol 1 as described (Corces et al., 2017) with some modifications. Viable cells (1x105) were resuspended in 50 μL cold ATAC-Resuspension Buffer (RSB) containing 0.1% NP40, 0.1% Tween-20 and 0.01% digitonin, and lysed for 3 min on ice. Lysates were washed with 1 mL cold ATAC-RSB containing 0.1% Tween-20 but no NP40 or digitonin. Samples were centrifuged for 10 min at 500×g at 4°C, the supernatants removed, and the pellets resuspended in 50 μL transposition mix (Tagment DNA buffer from Illumina, #15027866) containing 2.5 μL Tagment DNA TDE1 Enzyme (Illumina #15027865). After 45 min incubation at 37°C and 1000 rpm in an Eppendorf ThermoMixer, chromatin fragments were isolated using the Zymo Research DNA Clean & Concentrator kit (ZymoResearch ZY-D4013). For pre-amplification of transposed fragments, 5 PCR cycles were run using primers Ad1 and Ad2.x (Buenrostro et al., 2015). The remaining cycles of library amplification were determined by qPCR (Adams et al., 2000). Another clean-up was performed using the Zymo Research DNA Clean & Concentrator kit (21 µL elution volume) followed by size selection using AMPure XP beads (Beckman Coulter #A63880), with samples adjusted to 100 µL plus 55 µL of beads to remove large fragments. Samples were incubated for 5 min before bead separation using a magnetic stand and transfer of the supernatant to a new tube. Another 225 µL of beads were added followed by incubation for 10 min and separation on a magnetic stand. After washing with 80% ethanol, beads were resuspended in 20 μL elution buffer and incubated for 5 min. After separation on a magnetic stand, the eluate was transferred to a new tube