GSM5268031: WT_rep3_ATACseq; Mus musculus; ATAC-seq
The ATAC-seq experiment was performed according to the Omni-ATAC protocol previously published (Corces et al., 2017), with modifications. Freshly isolated murine female thymocytes were separated using Lympholyte®-M (Cedarlane, CL5030) according to the manufacturer's protocol, to ensure the presence of only viable cells. Isolated cells were washed twice with 1X PBS and aliquots of 10,000 cells were used for analysis. The cell pellet was gently resuspended by pipetting up and down three times in 50 µl of ice cold ATAC-RSB-NTD Buffer (10 mM Tris-HCl – pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween-20, 0.01% Digitonin) and incubated on ice for 3 minutes. Cell lysis was stopped by adding 1 ml of cold ATAC-RSB-T Buffer (10 mM Tris-HCl – pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20) and inverting the tube three times to mix. Nuclei were centrifuged at 1,000 g, at 4°C for 10 minutes. The pellet was resuspended in 50 µl of Transposition Mix [25 µl 2X TD buffer (20 mM Tris-HCl – pH 7.6, 10 mM MgCl2, 20% Dimethyl Formamide – before adding DMF, the pH was adjusted to 7.6 with 100% acetic acid), 2.5 µl transposase (100 nM final), 16.5 µl PBS, 0.5 µl 1% digitonin, 1 µl 5% Tween-20, 4.5 µl H2O] by pipetting up and down six times. The reaction was incubated at 37°C for 30 minutes with occasional pipetting. DNA was purified with a DNA Clean & Concentrator-5 Kit (Zymo Research, D4013) according to the manufacturer's protocol. DNA was eluted in 20 µl of Elution Buffer. All the material was used in a PCR reaction, together with 25 µl Phusion HF 2X Master Mix (NEB, M0531L) and 2.5 µl of each Nextera Index 1 (N7XX) and Nextera Index 2 (N5XX) primers from a Nextera DNA Sample Preparation Index Kit (Illumina, FC-121-1011). PCR was performed according to the following protocol: initial incubation at 72°C for 5 min and 98°C for 1 min, then 5x cycles of 98°C for 15 s, 63°C for 35 s and 72°C for 60 s. Samples were put on ice and 5 µl of the pre-amplified mixture was combined with 15 µl of qPCR Master Mix using the following set-up: 3.25 µl water, 0.5 µl primer FWD, 0.5 µl primer REV, 0.75 µl 20X SYBR Green, 5 µl Phusion HF 2X Master Mix, 5 µl pre-amplified sample. The qPCR reaction was run for additional 20 cycles following the program: initial incubation at 98°C for 1 min, then 20x cycles of 98°C for 15 s, 63°C for 35 s and 72°C for 60 s. Based on the Rn (Fluorescence) vs Cycle linear plot, a cycle with 1/4 up to 1/3 of the maximum fluorescence level was determined. This was the number of additional PCR cycles to run on the pre-amplified libraries which were stored on ice until this point. The final amplified libraries were purified using a DNA Clean & Concentrator-5 Kit (Zymo Research, D4013) according to the manufacturer's protocol and eluted in 20 µl of Elution Buffer. Libraries were quantified by Qubit and analyzed on a Bioanalyzer as previously described, followed by two-sided size selection using AMPure Beads. Libraries were sequenced on an Illumina® HiSeq 4000 2x 75 bp platform.