- Materials
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- 1.6 ml of whole blood
- Consumables
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- RBC Lysis Solution (QIAGEN, 158106)
- 10X phosphate-buffered saline (PBS), pH 7.4 (Thermo Fisher, 70011044)
- 15 ml Falcon tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
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- Temperature-controlled microfuge
- P1000 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
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PBMC sample preparation for the Ultra-long DNA experiment
Approximately 6 million isolated PBMCs must be prepared from 1.6 ml of whole blood to use as input in the Ultra-long DNA experiment.
Users may isolate PBMCs by any means they feel are most appropriate for the whole blood sample to be used. If 6 million cells have been isolated, users can start from day 1 of the Ultra-long DNA experiment.
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In a fresh 15 ml Falcon tube, prepare 10 ml of 1x PBS in nuclease-free water as follows:
Reagent Volume 10X PBS 1 ml Nuclease-free water 9 ml Total 10 ml -
Add 4.8 ml of RBC Lysis Solution to 1.6 ml of whole blood in a 15 ml Falcon tube.
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Gently invert the tube ten times to mix.
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Incubate for 5 minutes at room temperature and gently invert twice during the incubation.
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Centrifuge at 2000 x g for 2 minutes at 4°C to pellet the white blood cells.
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Discard the supernatant by pouring. There will be ~200 µl supernatant remaining in the tube.
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Resuspend the cells in the residual supernatant by gently flicking the tube.
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Make up the volume to 1.6 ml with 1x PBS.
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Repeat steps 1-7 twice more to complete three washes in total.
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After the final spin, remove the entire supernatant by pouring and aspirating any remaining supernatant.
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Resuspend the cell pellet in 40 µl 1x PBS. There will be approximately 6 million cells in the suspension.