SUPPORTING INFORMATION
Table S1. Normalized median values for Nile Red (NR) and chlorophyll (CHL)
fluorescence of mutants measured by flow cytometry, used to generate Figure 6.
Mutant/wild-type values for NR/CHL, NR and CHL are shown.
Table S2. Lipid species identified using MTQ-LC-MS and MS/MS analysis. The
columns represent the following values, respectively: (A) sample name, (B-AW)
lipid species area normalized to the internal standard triheptadecanoin, (AX)
summation of normalized peak areas.
Table S3. Fatty acid species identified by GC-FID analyses of wild-type and
mutants. The columns represent the following values, respectively: (A) sample
name, (B-P) lipid quanity in µg FAME per ml of culture, (Q) sum of FAME in µg
FAME per ml of culture.
Figure S1. Nile Red staining increases signal in the Nile Red channel but not in
the Chlorophyll channel. The data used to generate Figure 2e,f was re-plotted as
a scatter plot. Wild-type cells were treated with 0.011% Triton X-100 and stained
with 1 µg/ml Nile Red in methanol (1% final volume) or treated with methanol
alone (unstained sample).
Figure S2. Nile Red stained cells show a bimodal staining when no detergent is
added. Wild-type cells were stained with 1 µg/ml Nile Red in methanol (1% final
volume) or treated with methanol alone (unstained sample). No Triton X-100 was
present in either sample. Cells were analyzed by flow cytometry.
Figure S3. Addition of Triton X-100 levels of 0.014% or higher perturbs cellular
organization. C. reinhardtii cells were deprived of nitrogen for three days and
stained with Nile Red in the presence of 0.011% or 0.014% Triton X-100.
Figure S4. Nile Red signal alone, or Nile Red and Forward Scatter, do not
resolve sta1 from wild-type cells as effectively as Nile Red and chlorophyll. (a)
Histogram of the Nile Red channel of Nile Red stained wild-type and sta1 data
from Figure 4a,b. (b) Scatterplot of Forward Scatter vs. Nile Red channels of the
data from Figure 4a-d.
Figure S5. The FACS procedure decreases colony recovery five-fold. CMJ030
wild-type cells were nitrogen-deprived, and either plated directly or stained and
passed through the FACS machine (set to collect all events). The % of initial cells
that gave rise to a colony was plotted. Error bars represent the span of %
recovery for two biological replicates.
Figure S6. Fatty acid lipidomics reveals increased total fatty acids and altered
fatty acid profiles of the mutants. (a) Total fatty acid content was determined by
GC-FID from the same samples as analyzed by LC-MS in Figure 7. Bars indicate
the mean of three biological replicates and error bars indicate standard error of
the mean. (b) The relative abundance of each fatty acid species in each mutant
is shown as a fold difference in percentage composition relative to average wildtype (colored boxes) and as a percentage of total fatty acid content (circles). The
fold difference panels represent one replicate each. The size of each circle is
proportional to the percentage of total fatty acid represented by the
corresponding fatty acid species, averaged over three biological replicates. Grey
square in WT2 indicates trace amount detected.
Figure S7. Southern Blot analysis of 10 mutants indicates that each mutant
contains one mutagenic cassette insertion. (a) 5 μg of genomic DNA from 10
mutants and their background strain (“WT”) was digested with StuI and run on a
0.7% TBE agarose gel. (b) Southern Blot from the gel in (a) was performed with
a probe against the AphVIII resistance marker gene. Both images are to scale.
Figure S8. Original image of 28 isolated mutants spotted on agar and stained
with iodine vapors. Individual spots from this image were cropped and
rearranged to produce Figure 6d. Mutants 25-28 were not suitable for FACS due
to extensive cell clumping and were not further analyzed.