Ab1127

After treatment with compounds, DNA was automatically purified using the Agencourt DNAdvance Genomic DNA Isolation Kit and an automatic dispenser (Biomek i5 or NXp, Beckman Coulter) according to the manufacturer’s instructions. Briefly, the cells were washed 3 times with PBS and incubated with 200 μl of Lysis buffer containing 50 mM dithiothreitol (DTT) and proteinase K at 55 °C for 1 h with constant agitation. The lysates were transferred to 96-well 1.2-ml deep well storage plates (Thermo, AB1127) and mixed with 100 μl of Bind1 buffer, and then the 170 μl of Bind2 buffer was then added, and the samples were again mixed. After collection of the DNA-binding magnetic beads, the beads were washed twice with 340 μl of 70% ethanol. DNA was eluted with 200 μl of MilliQ water.

RNA purification was automatically performed using Agencourt RNAdvance Cell v2 and an automatic dispenser (Biomek i5 or NXp, Beckman Colter) according to the manufacturer’s instructions. The cells were washed 3 times with PBS and then incubated with 63 μl of Lysis buffer containing proteinase K at 25 °C for 30 min and stored at -80 °C. After thawing and centrifugation at 1,000xg for 15 s, 175 μl of Binding buffer solution was mixed with the lysates in a 96-well 1.2-ml deep-well storage plate (Thermo, AB1127). The RNA-binding magnetic beads were washed with 200 μl of Wash buffer followed by 200 μl of 70% ethanol and then incubated with 5 U DNaseI (Nippon gene) at 25 °C for 15 min. The beads were then washed once with 138 μl of Wash buffer and twice with 200 μl of 70% ethanol. RNA was eluted with 40 μl of MilliQ water (RNase and DNase free).

Immunoprecipitation of HLA molecules was performed using the Agilent AssayMap instrument as described before. Briefly, 100 µg of biotinylated anti-pan HLA class I (W6-32, produced in house) were immobilized on streptavidin cartridges (Agilent, G5496-60010) by passing over the cartridge at 5 µL/minute and washing three times with PBS. Cell lysates were thawed and filtered using a 0.2 µm hydrophilic filter plate (Analytical Sales & Services #96432-10) and loaded in a 96 well polypropylene plate (Thermo Scientific #AB1127). Filtered lysate was passed through the antibody loaded cartridges at 5 µL/minute at room temperature. Following that, the cartridges were washed twice with 50 mL of 100 mM ammonium acetate and once with 50 µL of water at 25 µL/minute. The HLA:peptide complexes were eluted with 50 µL of 5% acetic acid in 0.1% TFA at 2 µL/minute. Eluted samples were centrifuged in 10K MWCO spin filters (MilliporeSigma #MRCPRT010) equilibrated with BSA, angiotensin-I and acetic acid. 20 µL of the filtered samples were loaded in a 96-well polypropylene PCR plate. From this volume, 18 µL of isolated peptides were injected into the LC-MS system. Notably, the injection volume corresponds to the peptides originating from an estimated 3.6 million cells.