Materials and reagents

Biological material

1. We used Chlamydomonas reinhardtii D66 (CC-4425) [8] and CLiP library (CC-4533) [9] strains, but any strain could be used

Reagents

Note: Unless specified otherwise, the reagent can be stored at room temperature.

1. TAP (tris-acetate-phosphate) solution (Life Technologies, catalog number: T8050)

2. S-nitrosoglutathione (GSNO) produced in our laboratory but also available for purchase (Sigma, catalog number: N4148), stored at -20 °C

3. Evans blue powder (Sigma, catalog number: E2129)

4. Phenol:chloroform:isoamyl (25:24:1) (Sigma, catalog number: 77617), stored at 4 °C

5. Ethanol 100% (Sigma, catalog number: 32205-M)

6. Trizma^® base (Sigma, catalog number: 93350)

7. Hydrochloric acid (HCl) (Sigma, catalog number: 258148)

8. Sodium chloride (NaCl) (Sigma, catalog number: S7653)

9. Ethylenediaminetetraacetic acid tetrasodium (EDTA) (Sigma, catalog number: E6511)

10. Sodium dodecyl sulfate (SDS) 20% (Sigma, catalog number: 05030)

11. Sodium acetate (Sigma, catalog number: S2889)

12. RNase A, 10 mg/mL (Thermo Scientific, catalog number: EN0531), stored at -20 °C

13. Agarose (Sigma, catalog number: A9539)

14. Tris borate EDTA (TBE) buffer (Sigma, catalog number: T4415)

15. Ethidium bromide solution (Sigma, catalog number: 46067)

16. GeneRuler 100 bp Plus DNA ladder (Thermo Scientific, catalog number: SM0321), store at 4 °C

17. DAF-FM diacetate (4-amino-5-methylamino-2’,7’-difluorescein diacetate) (Invitrogen, catalog number: D-23844), store at -20 °C, protected from light

1. 50 mM GSNO solution (see Recipes)

2. DNA extraction buffer (see Recipes)

3. Sodium acetate 3.3 M (see Recipes)

4. TBE 0.5× solution (see Recipes)

5. Evans blue solution (see Recipes)

1. 50 mM GSNO solution

   1. Weigh 20 mg of GSNO and dissolve it in 800 µL of TAP medium [10] in a 1.5 mL tube.

   2. Verify the concentration of the 100-fold diluted solution using the molar extinction coefficient of GSNO (922 M^-1·cm^-1 at 335 nm) [11].

   Note: It is better to prepare fresh GSNO for each experiment to avoid its oxidation.




2. DNA extraction buffer (for 500 mL)

   Reagent	Final concentration	Amount
   Tris-HCl (1 M, pH 7.5)	200 mM	100 mL
   NaCl (1 M)	250 mM	125 mL
   EDTA (0.5 M, pH 8)	25 mM	25 mL
   SDS (20%)	0.5%	12.5 mL
   MilliQ water	n/a	262.5 mL
   Total	n/a	500 mL

   Note: It is recommended to autoclave extraction buffer (add SDS after autoclaving) before proceeding to DNA extraction.




3. Sodium acetate 3.3 M (for 100 mL)

   Reagent	Final concentration	Amount
   Sodium acetate	3.3 M	27 g
   MilliQ water	n/a	100 mL
   Total	n/a	100 mL

   1. Dissolve using a magnetic stirrer and stir bar.

   2. Filter sterilize (0.22 µm).




4. TBE 0.5× solution

   Reagent	Final concentration	Amount
   TBE (10×)	0.5×	100 mL
   MilliQ water	n/a	1.9 L
   Total	n/a	2 L




5. Evans blue solution

   Reagent	Final concentration	Amount
   Evans blue powder	0.5 %	0.5 g
   TAP	n/a	100 mL
   Total	n/a	100 mL

   Filter sterilize (0.22 µm).

Laboratory supplies

1. Spectrophotometer semi-micro cuvette (Biosigma, catalog number: BSA002)

2. 24-well plates (Evergreen Labware, Medical Caplugs, catalog number: 222-8044-01F)

3. Surgical tape (Micropore^TM, catalog number: 1530-0)

4. Counting chamber Neubauer (Blaubrand, catalog number: BR717805)

Equipment

1. Spectrophotometer (Implen Nanophotometer)

2. Microplate reader (ClarioStar Plus, BMG LABTECH)

3. Orbitron Rotary shaker (Infors)

4. Olympus BX43 microscope

5. Thermomixer (Eppendorf, catalog number: 5382000015)

6. Vortex (Dutsher Vortex Genie 2, catalog number: 079008)

7. Electrophoresis tanks (Embitec^® runOne^TM, model: EP-2000)

8. Camera (Q-IMAGING Micropublisher 3.3 RTV)

9. NanoDrop One (Thermo Scientific)

10. GelDoc Go Imaging System (Bio-Rad)

Software and datasets

1. Image Lab v6.1 for the GelDoc Go Imaging System (Bio-Rad)

2. Smart control data analysis for ClarioStar Plus (MARS, BMG LabTech)

Procedure