GSM4859448: Control HSC rep3 [ATAC-seq]; Mus musculus; ATAC-seq
Sorted 5,000 CD45.2+Lin-Sca1+cKit+CD34-Flt3-CD150+ were washed with 400 μl of resuspension buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2), and lysed in 50 μl of resuspension buffer supplemented with 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin for 3 min on ice. Nuclei were washed with 1 ml of resuspension buffer supplemented with 0.1% Tween-20, and then 50 μl of transposition mix (16.5 μl PBS, 5 μl water, 25 μl 2× TD Buffer, 2.5 μl Tn5 enzyme (Illumina), 0.5 μl 1% digitonin, and 0.5 μl 10% Tween-20) was added to the cell pellet. The samples were incubated for 30 min at 37 °C. Reactions were cleaned up with MinElute Reaction Cleanup Kit (QIAGEN) and eluted by 12 μl of EB buffer. Fragmented DNA were amplified by NEBNext Ultra II Q5 with ATAC primers for five cycles. Additional PCR cycles were evaluated by real-time PCR. Final product was cleaned with Ampure XP Beads (Beckman Coulter).