Am9759

The assembly of the Tn5 and T7-Tn5 transposases were performed as described (Picelli et al. 2014 (link)). Briefly, oligonucleotides (T7-Tn5ME, Tn5MErev, Tn5ME-A, Tn5ME-B) were resuspended in water to a final concentration of 100 µM each. Equimolar amounts of Tn5MErev/Tn5ME-T7, Tn5MErev/Tn5ME-A, and Tn5MErev/Tn5ME-B were mixed in separate 200-µL PCR tubes. These oligos mixtures were denatured on a thermocycler for 5 min at 95°C and cooled down slowly on the thermocycler by turning off the thermocycler. The T7-Tn5 transposase was assembled with the following components: 0.25 vol Tn5MErev/Tn5ME-T7 (final concentration of each double-strand oligo is now 50 µM each), 0.4 vol glycerol (100% solution), 0.12 vol 2× dialysis buffer (100 mM HEPES-KOH at pH 7.2, 0.2 M NaCl [Invitrogen AM9759], 0.2 mM EDTA [Invitrogen AM9290G], 2 mM DTT, 0.2% Triton X-100 [Sigma-Aldrich T8787] 20% glycerol [Sigma-Aldrich G9012-500]), 0.1 vol SL-Tn5 (50 µM), 0.13 vol water. The reagents were mixed thoroughly but gently, and the solution was left on the bench for 1 h at room temperature to allow annealing of oligos to Tn5. The Tn5 transposase was assembly with same procedure as T7-Tn5 transposase but with following oligos: 0.25 vol Tn5MErev/Tn5ME-A and 0.25 vol Tn5MErev/Tn5ME-B.

Aliquots of nuclei with epitope retrieval from human GBM or human CRC samples (100 000 cells/0.5 ml Qubit tube (Invitrogen, Q32856)) were washed once with FACT-seq Antibody buffer (20 mM HEPES(K+) pH 7.6 (HEPES: Sigma-Aldrich, H3375; KOH: Sigma-Aldrich, 484016), 150 mM NaCl (Invitrogen, AM9759), 2 mM EDTA (Invitrogen, AM9260G), 0.5 mM spermidine (Sigma-Aldrich, S2626), 0.05% digitonin (Millipore, 300410), 0.01% IGEPAL^® CA-630 (Sigma-Aldrich, 13021-50), 1× protease inhibitors (Sigma-Aldrich, 11873580001), 1% BSA (Miltenyi Biotech MACS, 130-091-376)). H3K27ac FACT-seq sequencing libraries were prepared with same methods as in the mouse samples.

For FFPE-ATAC samples, single nuclei were isolated following the nuclei isolation protocol stated in the section on nuclei isolation from FFPE tissue sections. For frozen samples, nuclei were isolated following the nuclei isolation protocol in the section on standard ATAC-seq on frozen tissue. For genomic DNA purification, 1 million isolated nuclei were spined down at 3000g for 10 min and then resuspended with 100 µL of lysis buffer (50 mM Tris-HCl at pH 7.5 [Invitrogen 15567027], 1 mM EDTA [Invitrogen AM9260G], 1% SDS [Invitrogen 1553-035], 200 mM NaCl [Invitrogen AM9759], and 200 µg/mL Proteinase K [Thermo Fisher Scientific EO0491]). Nuclei suspension was incubated overnight at 65°C with 1200 rpm shaking in a heat block. On the next day, the mixture was purified with a Qiagen MiniElute purification kit (Qiagen 28004) and eluted in 20 µL of elution buffer. Purified genomic DNA was measured and was run on a 1.5% agarose gel (Lonza 50004) to check size distribution.