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The Peter Doherty Institute for Infection and Immunity, University of Melbourne

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Namespace:The Peter Doherty Institute for Infection and Immunity, University of Melbourne

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Protocols: Viable PbTII cells were FACS sorted from pooled splenic MACS-enriced CD4+ T cells. Raw ATAC-seq reads were mapped to mouse genome MGSCv37 (mm9) using BWA-MEM. Sorted cells were resuspended in 50 μl tagmentation mix (33 mM Tris-acetate, pH 7.8, 66 mM potassium acetate, 10 mM magnesium acetate, 16% dimethylformamide (DMF), 0.01% digitonin and 5 μl of Tn5 from the Nextera kit from Illumina, Cat. No. FC-121-1030). Cell/ tagmentation mixture was then incubated at 37°C, 800rpm on an Eppendorf thermomixer for 30 minutes. Tagmentation reaction was stopped by the addition of equal volume (50 μl) of stop buffer (10 mM Tris-HCl, pH 8.0, 20 mM EDTA, pH 8.0) followed by incubation in ice for 10 minutes. 150 uL of DPBS/ 0.5% BSA was added to the mixture and nuclei suspension was then transferred to a FACS tube. 0.0006% DAPI (in-house) was added for staining the nuclei prior to single-nuclei sort into 384-well plate (Eppendorf, Cat. No. 0030128508) containing 2 µl of 2X lysis buffer (100 mM Tris.HCl, pH 8.0, 100 mM NaCl, 40 µg/ml Proteinase K (New England BioLabs, P8107S), 0.4% SDS. Single-nuclei lysates were sealed and spun at 100 x g for 1 minute then immediately frozen on dry ice and stored at -80°C. Tn5 release and proteinase K digestion was then performed on a PCR machine (MJ Research Peltier Thermal Cycler) at 65°C for 15 minutes followed by the addition of 2 µl of 10% Tween to quench the SDS. 5 µl of NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs Inc, Cat. No. M0541L) was then added to a pre-prepared 384-well plate (Eppendorf, Cat. No. 0030128508) containing 1 µl of 10 µM i5 and i7 indexing primer mix (5 µM each) (Integrated DNA Technologies). The indexing primers were arranged in a combinatorial manner to allow multiplexing of up to 1536 (4x 384-well plates) sorted nuclei into one sequencing pool. The plate containing the PCR Master Mix and indexing primers was stamped onto the single nuclei, mixed briefly, sealed and spun at 100 x g for 1 minute. Amplification was performed on a PCR machine (MJ Research Peltier Thermal Cycler) with 72°C 10 min, 98°C 5 min, 20 cycles of [98°C 10 s, 63°C 30 s, 72°C 20 s]. The 384-well plate containing the PCR product was inverted into a VBLOK200 reservoir (Clickbio Cat. No. CBVBLOK200-1) and spun at 200 g for 1 minute. The combined reactions were then transferred to a 5 ml Eppendorf tube, (Eppendorf, Cat No. 0030119401) and the DNA purified, and size selected with an AMPure XP workflow (Beckman Coulter, Cat No. A63880).