Facs canto

For 5-ethynyl-2′-deoxyuridine (EdU) incorporation, cells were stained according to the manufacturer’s instructions using an EdU Staining Proliferation Kit iFluor 488 (ab219801, Abcam). For cell cycle analysis, the cells were collected using trypsin and suspended in Hanks balanced salt solution buffer with 2% FBS, then washed twice with ice-cold PBS. The cells were then incubated with 100 μg/mL RNase and 100 μg/mL propidium iodide (PI) for 15 min and analyzed using FACS Canto (Beckman Coulter, Brea, CA, USA). Single cells were gated and analyzed to differentiate between G0/G1, S, G2 + M phases, and sub-G0/G1 using FCS Express 6 Plus (De Novo software, Los Angeles, CA, USA). Cell death was measured using annexin V and PI staining. The cells were washed with ice-cold PBS and suspended in 100 μL of staining solution containing 5 μL of annexin V-Alexa-488-conjugate. After 15 min of incubation at room temperature, 400 µL of fresh binding buffer was added per sample containing 1 μL of 20 μg/mL PI, then immediately analyzed using the FACS Canto instrument (Beckman Coulter).

ATL patient PBMCs were thawed and washed with PBS containing 0.1% BSA. To discriminate dead cells, we used the LIVE/DEAD Fixable Dead Cell Stain Kit (Invitrogen). For cell surface staining, cells were stained with APC anti-human CD4 (clone: RPA-T4) (BioLegend) and anti-SynCAM (TSLC1/CADM1) mAb-FITC (MBL) antibodies for 30 minutes at 4°C followed by a wash with PBS. HTLV-1 infected cells (CADM1+ and CD4+) were sort-purified with FACS Canto (Beckman Coulter) to reach 98–99% purity. Data was analyzed by FlowJo software (Treestar). Soon after the sorting, 10000-50000 HTLV-1 infected cells were centrifuged and used for ATAC-seq as previously described [5 (link)]. Total RNA was isolated from the remaining cells using the RNeasy Mini Kit (Qiagen). Library preparation and high-throughput sequencing were performed by Macrogen Inc. (Seoul, Korea). The diagnostic criteria and classification of clinical subtypes of ATL were performed as previously described [28 ]. 77 ATAC-seq datasets from 13 human primary blood cell types and datasets from 42 AML patients were obtained from the Gene Expression Omnibus (GEO) with accession number GSE74912 [7 (link)]. ATAC-seq datasets from 7 CLL patients were obtained from GSE111015 [18 (link)] and RNA-seq datasets of CD4⁺T and Mono cells were obtained from GSE74246 [7 (link)].