GSM8829108: MEFs, 24h iMPC medium, rep2; Mus musculus; ATAC-seq
For ATAC-Seq, the protocol modifications of the Kestner Lab of the Omni-ATAC protocol were used (Ackermann et al., 2016; Corces et al., 2017). Cells were washed 1x with PBS and collected by incubating them with Accutase cell detachment solution at 37°C in 5% CO2 for 7 minutes. After inactivation with iMPC medium, cells were centrifuged and resuspended in sterile-filtered 2% BSA (Albumin (BSA) Fraction V, (pH 7.0)) in PBS. 50k cells per condition were transferred into 1.5mL Protein Lo-Bind tube. Cells were then spun down at 500g for 5 minutes at 4°C, washed with ice-cold PBS, spun down again with the same settings and resuspended in nuclei isolation buffer containing 10mM Tris-HCl (pH 7.5), 10mM NaCl, 3mM MgCl2, 0.1% Igepal, 0.1% TWEEN 20 and 0.01% Digitonin. After incubation for 3 minutes on ice, wash buffer containing 10mM Tris-HCl (pH 7.5), 10mM NaCl, 3mM MgCl2 and 0.1% TWEEN 20 was added to the nuclei and the samples were centrifuged at 500g at 4°C for 10 minutes. Each nuclei preparation was then resuspended in transposition reaction mix containing 25µL 2x Tagment DNA buffer (Illumina, 20034197), 16.5µL PBS, 0.5µL 10% TWEEN 20, 0.5µL 1% Digitonin, 2.5uL TDE1 Tagment DNA Enzyme (Illumina, 20034197) and 5µL nuclease-free H2O. The samples were then incubated at 37°C for 30mins at 1,000rpm. Tagmented DNA was then isolated and purified using MinElute Reaction Cleanup Kit according to manufacturer's protocol and eluted in 10µL. The purified transposed DNA was then amplified for 12 cycles using NEBNext High-Fidelity 2X PCR Master Mix and the custom Nextera PCR primers Ad1_noMX and Ad2.1-6 indexing primers at 1.25µM from Buenrostro et al., 2013. The amplified libraries were then purified from primers with a single left-side 1.8x AMPure XP Beads-Based Reagent beads purification. Samples were then eluted in 22µL elution buffer and a 1:15 dilution was analyzed on the 4200 TapeStation System (Agilent Technologies) using the D5000 High Sensitivity ScreenTapes and reagents.