Table 1

Summary of extraction protocolsa

Treatment name in Fig. 3Summary of treatmentb
NaOH leach2 20-min leaches with 160 μl 0.25 M NaOH, neutralization with HCl; centrifugation, removal of the supernatant, dilution with 500 μl Tris
Lyso/n.ases190 μl lysis buffer, sonication, lysozyme, DNase and RNase; centrifugation, removal of the supernatant, addition of 300 μl Tris, centrifugation, removal of the supernatant
Lysozyme190 μl lysis buffer, sonication, lysozyme; centrifugation, removal of the supernatant, addition of 300 μl Tris, centrifugation, removal of the supernatant
Freeze-thawFrozen pellets thawed at room temp, addition of 200 μl Tris, vortexing, centrifugation, removal of the supernatant; addition of 300 μl Tris, centrifugation, removal of the supernatant
Dry boiledSonication of the cell pellet, boiling, sonication; addition of 200 μl Tris, centrifugation, removal of the supernatant; addition of 300 μl Tris, centrifugation, removal of the supernatant
Tris boiledAs for “dry boiled,” but addition of the 200 μl Tris before sonication and boiling
Dry boil/lysoSonication of the pellet, boiling, sonication, addition of 190 μl lysis buffer and lysozyme; centrifugation, removal of the supernatant; addition of 300 μl Tris, centrifugation, removal of the supernatant
NaOH boiled160 μl 0.25 M NaOH, sonication, boiling, sonication; centrifugation, removal of the supernatant, neutralization with HCl; addition of 250 μl Tris, centrifugation, removal of the supernatant
Short Tris boilAs for “Tris boiled,” but only 2 min boiling
Long Tris boilAs for “Tris boiled,” but 20 min boiling
Nucleases after boiling200 μl lysis buffer, sonication, boiling, sonication, DNase and RNase; centrifugation, removal of the supernatant; addition of 300 μl Tris, centrifugation, removal of the supernatant
Lysozyme & nucleases after boiling200 μl lysis buffer, sonication, boiling, sonication, lysozyme, DNase and RNase; centrifugation, removal of the supernatant; addition of 300 μl Tris, centrifugation, removal of the supernatant
Proteinase K after boiling200 μl Tris, sonication, boiling, sonication, proteinase K treatment for 10 min at 37°C; centrifugation, removal of the supernatant; addition of 300 μl Tris, centrifugation, removal of the supernatant
All enzymes after boiling200 μl lysis buffer, sonication, boiling, sonication, lysozyme, DNase and RNase; proteinase K treatment for 10 min at 37°C; centrifugation, removal of the supernatant; addition of 300 μl Tris, centrifugation, removal of the supernatant
EDTAAs for “Tris boiled,” but 10 or 50 mM EDTA was added to buffer before boiling for 10 min
Proteinase K for VineyardAs for “Proteinase K after boiling,” but boiled for 10 min and treated with proteinase K for 1 h
EDTA proteinase KAs for “Proteinase K for Vineyard,” but 10 or 50 mM EDTA was added to buffer before boiling
  • a Centrifugation was always for 1 min at 16,100 × g, and sonication was always for 15 s. Boiling was for 5 min for Synechococcus (Fig. 3a and b) and 10 min for Vineyard Sound samples (Fig. 3c).

  • b Lysis buffer, 10 mM Tris (pH 7.0), 100 mM NaCl, 2 mM MgCl2, and 2 mM EDTA; lysozyme, 10 μl of 25 mg ml−1 lysozyme, incubation for 10 min at 37°C; DNase and RNase, 10 units DNase, 100 units RNase T1, and 2.5 units RNase A, incubation for 10 min at 37°C; proteinase K, 10 μl of 20 mg ml−1 proteinase K.