PD-1 instructs a tumor suppressive metabolic program to restrain AP-1 activity in T cell lymphoma: Sample S17
50,000 malignant cells from CTCL patients were sorted by FACS. The cells were pelleted and resuspended in lysis buffer (10 mM Tris HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40 [11332473001, Roche], 0.1% Tween-20 [P1379, Sigma], and 0.01% digitonin [G944A, Sigma]). Lysis was performed on ice for 3 min, before washing with 10 mM Tris HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2 buffer containing 0.1% Tween-20. The nuclei were pelleted, and transposition (Tagment DNA Enzyme I, 15027865, Illumina) was performed at 37 °C for 30 min with shaking at 1,000 rpm in 10 mM Tris-HCl pH 7.6, 5 mM MgCl2, 10% dimethyl formamide (D4551, Sigma), PBS, 0.1% Tween-20, 0.01% digitonin. DNA was column purified (T1030L, NEB), and the transposed DNA was amplified in 50 μl reaction volumes with a modified version of custom-designed primers. The reaction was monitored after 4 cycles with qPCR using 5 μl PCR reaction mixture and the same primers in 15 μl total volume (KK4617, Roche). Cycles were added as calculated, then the amplified samples were purified, and size selected using SpeedBeads (GE65152105050250, Sigma) in 22% PEG. Libraries were quality-controlled using Qubit and Bioanalyzer (5067-4626, Agilent), and 76 bp paired-end sequencing was performed on an Illumina NextSeq 500.