GSM8322476: Inflamed_basal prostate stem cells_2; Mus musculus; ATAC-seq
Minced prostate tissues from naïve or inflamed POET3 mice were digested with collagenase followed by trypsinization. Dissociated cells were passed through 20G needles and 40 µm cell strainers to eliminate aggregates, followed by removal of red blood cells by ACK buffer. Isolated cells were stained with fluorescence-conjugated specific antibodies for CD45, CD31, Sca-1 and CD49f. CD45-CD31-Sca-1+CD49f+ basal stem cells were sorted on the BD FACSAria under sterile conditions. Nuclei were prepared by cell lysis using ice cold lysis buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% IGEPAL CA-630) before immediate transposition of DNA in 25 μl 2x TD buffer, 2.5 μl Transposase (Illumina) and 22.5 μl of nuclease free water for 30 minutes at 37 degree. Samples were then purified using Mini Elute Kit (Qiagen). PCR amplification and library prep were performed using 10 μl transposed DNA, 9.7 μl nuclease free H2O, 0.3 μL 100x SYBR Green I (Invitrogen S-7563), 25 μL NEBNext High-Fidelity 2x PCR Master Mix (New England Labs M0541), and 2.5 μl each of individual 25μM dual unique Nextera PCR primers in a 50 μl reaction.