GSM6256807: mMDSC, Male, Healthy, Biological replicate 2; Mus musculus; ATAC-seq
Cells were sorted from the bone marrow of sham-injected or GL261 cell-implanted male and female C57BL/6 mice. Cells were washed with cold PBS and counted on a Countess (Invitrogen). Pellets were resuspended in 1 ml of nuclei permeabilization buffer (PBS with 1 mM dithiothreitol, 5% bovine serum albumin, 0.2% IGEPAL-CA630 (Sigma Aldrich) and 1x cOmplete EDTA-free protease inhibitor (Roche)) and rotated for 10 min at 4°C as per the Ren et al. protocol (https://www.encodeproject.org/documents/4a2fc974-f021-4f85-ba7a-bd401fe682d1/). The nuclear suspension was filtered on a 30 µm CellTrics (Sysmex) and spun at 500 x g for 5 min at 4°C. The resulting pellet was resuspended in 50 µl tagmentation buffer (33 mM Tris-acetate pH 7.8, 66 mM potassium acetate, 11 mM magnesium acetate, and 16% N'N-dimethylformamide, in water). An aliquot of nuclear suspension was counted on a Countess. For each tagmentation reaction, 160,000 nuclei were mixed with 1 µl of Tagment DNA enzyme in a total of 20 µl and incubated for 30 min with 500 rpm at 37°C. Then 200 µl buffer PB (Qiagen) and 10 µl sodium acetate (3M, pH 5.2) were added, and the sample was purified using a MinElute PCR purification kit (Qiagen) and eluted in 10 µl EB. Fragments were amplified by PCR using indexed primers, then purified using the MinElute PCR purification kit with elution in 40 µl EB. Size selection was performed with PCRclean Dx beads (ALINE) by adding 160 µl EB to the 40 µl sample and adding 110 µl beads. After mixing and incubation for 5 min at room temperature, the tube was put on a magnetic stand and the supernatant transferred to a new tube, to which 190 µl beads was added. This suspension was mixed, incubated, and put on the magnetic stand, and this time the supernatant was discarded.  Beads were washed twice with 70% ethanol, then the DNA was eluted from the beads with 20 µl EB. The indexed libraries were sequenced on a NextSeq high-output flow cell, paired-end, 75 cycles.