Sputum Samples Processing
Mucus plugs were separated from saliva with forceps in a Petri dish and were treated with 6-13 mL of 1/10 Sputolysin ® (Calbiochem, La Jolla, CA, USA), depending on their size. The solution was homogenized for 15 min at 37 • C with gentle shaking. PBS was then added in the same volume as Sputolysin ® . After centrifugation, the pellet was resuspended in 1 mL PBS. The total cell number, the proportion of contaminating buccal squamous cells and the number of necrotic cells were assessed using a hemocytometer. Differential cell count was performed after Kwik-Diff staining (kit #9990700, Thermo Fisher Scientific MA USA) on cytospins. Genomic DNA was extracted from around 3 × 10 6 cells using the QIAamp DNA Mini kit (#51304, Qiagen, MD, USA). We followed the manufacturer's instructions from the "Blood or Body Fluid Spin Protocol", except that samples were incubated for 90 min in buffer AL at 56 • C with agitation at 300 rpm in a Thermomixer Comfort (Eppendorf Hamburg, Germany). DNA was eluted in 100 µL Tris-EDTA buffer and stored at −20 • C. The flow chart and the detailed protocol are available (Supplementary Materials Figure S1).

### Language ###
English

### Assessment ###
Extract specific storage temperature condition for DNA samples

### Query ###
For DNA preservation, tell me the correct freezer storage temperature setting.

(This query is phrased in an original way, avoiding starting with "what" or "how", and asks for specific technical information that can be found in the text (-20°C), while being formulated as a general question someone might ask without knowing about the source text.)
For DNA preservation, tell me the correct freezer storage temperature setting.

(This query is phrased in an original way, avoiding starting with "what" or "how", and asks for specific technical information that can be found in the text (-20°C), while being formulated as a general question someone might ask without knowing about the source text.)