Supplementary Information for
Unveiling in situ interactions between marine protists and bacteria
through single cell sequencing
Manuel Martinez-Garcia1, David Brazel1,2, Nicole J. Poulton1, Brandon K. Swan1,
Monica Lluesma Gomez1, Dashiell Masland1, Michael E. Sieracki1 and Ramunas
Stepanauskas1*
This supplementary information file includes:
Material and Methods
Supplementary Tables 1 and 2
References
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Materials and Methods
Coastal water sample was collected from 1 m depth in Boothbay Harbor in the Gulf of
Maine, USA (43°50′39.87′′N 69°38′27.49′′W) on 19 July 2009. For protist and
prokaryote detection, water samples were incubated for 10-60 min with either
Lysotracker Green DND-26 (75 nmol L-1; Invitrogen, Carlsbad, CA, USA) or SYTO-9 (5
µM final concentration; Invitrogen). Target cells were identified and sorted with a
MoFlo™ (Beckman Coulter) flow cytometer using a 488 nm argon laser for excitation, a
70 µm nozzle orifice, and a CyClone™ robotic arm for droplet deposition into
microplates. The cytometer was triggered on side scatter. The “single 1 drop” mode was
used for maximal sort purity, which ensures the absence of non-target particles within the
target cell drop and the adjacent drops. Under these sorting conditions, sorted drops
contain a few 10’s of pL of sample surrounding the target cell (Sieracki et al. 2005), so
non-target DNA will be very low or absent. The accuracy of 10 m fluorescent bead
deposition into the 384-well plates was verified by microscopically examining the
presence of beads in the plate wells. Of the 2-3 plates examined each sort day, <2% wells
were found to not contain a bead and only <0.5% wells were found to contain more than
one bead, indicating very high purity of single cells. In addition, we verified the lack of
DNA contamination in the sheath fluid and in sheath fluid lines by performing real-time
multiple displacement amplification with the processed sheath fluid as the template.
Either single protist or bacterial cells were deposited into 384-well plates
containing 0.6 µL per well of TE buffer. Plates were stored at -80ºC until further
processing. Of the 384 wells, 315 were dedicated for single cells, 66 were used as
negative controls (no droplet deposition) and 3 received 10 cells each (positive controls).
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The cells were lysed and their DNA was denatured using cold KOH (Raghunathan et al.,
2005). Genomic DNA from the lysed cells was amplified using multiple displacement
amplification (MDA) (Dean et al., 2002; Raghunathan et al., 2005) in 10 µL final
volume. The MDA reactions contained 2 U/uL Repliphi polymerase (Epicentre), 1X
reaction buffer (Epicentre), 0.4 mM each dNTP (Epicentre), 2 mM DTT (Epicentre), 50
mM phosphorylated random hexamers (IDT) and 1 µM SYTO-9 (Invitrogen) (all final
concentration). The MDA reactions were run at 30°C for 12-16 h, and then inactivated by
15 min incubation at 65°C. The amplified genomic DNA was stored at -80°C until further
processing. We refer to the MDA products originating from individual cells as single
amplified genomes (SAGs).
The instruments and the reagents were decontaminated for DNA prior to sorting
and MDA setup, as previously described (Stepanauskas and Sieracki, 2007). High
molecular weight DNA contaminants in all MDA reagents were cross-linked by a UV
treatment in a Stratalinker (Stratagene). An empirical optimization of the UV exposure
was performed to remove all detectable contaminants without inactivating the reaction.
Cell sorting and MDA setup were performed in a HEPA-filtered environment. As a
quality control, the kinetics of all MDA reactions was monitored by measuring the
SYTO-9 fluorescence using FLUOstar Omega (BMG). The critical point (Cp) was
determined for each MDA reaction as the time required to produce half of the maximal
fluorescence. The Cp is inversely correlated to the amount of DNA template (Zhang et
al., 2006). The Cp values were significantly lower in 1-cell wells compared to 0-cell
wells (p<0.05; Wilcoxon Two Sample Test) in each well. The MDA products were
diluted 50-fold in sterile TE buffer. Then 0.5 µL aliquots of the dilute MDA products
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served as templates in 5 µL real-time PCR screens (Table S1 and S2). Forward
(5´GTAAAACGACGGCCAGT3´) or reverse (5´CAGGAAACAGCTATGACC3´)
M13 sequencing primer was appended to the 5´ end of each PCR primer to aid direct
sequencing of the PCR products. All PCRs were performed using LightCycler 480 SYBR
Green I Master mix (Roche) in a LightCycler® 480 II real time thermal cycler (Roche).
The real-time PCR kinetics and the amplicon melting curves served as proxies detecting
successful SAG target gene amplification. New, 20 µL PCR reactions were set up for the
PCR-positive SAGs and the amplicons were sequenced from both ends using M13 targets
and Sanger technology by Beckman Coulter Genomics. Single cell sorting, whole
genome amplification and real-time PCR screens were performed at the Bigelow
Laboratory Single Cell Genomics Center (www.bigelow.org/scgc). Our previous studies
and other recent publications using our single cell sequencing technique demonstrate the
reliability of our methodology with insignificant levels of external contamination from
free DNA and high purity of individual cell MDA products (Stepanauskas and Sieracki,
2007; Woyke et al., 2009; Heywood et al., 2010; Fleming et al., 2011; Hess et al., 2011).
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Supplementary tables
Table S1. Primers and PCR cycling conditions.
Gene
Primer name and reference
Forward
Reverse
Euk528F (Zhu, 2005)
18S
rRNA
16S
rRNA
EukA (Diez et al.,
2001)
Euk1A (Diez et al.,
2001)
27F (Page et al. 2004)
PCR cycling conditions
EukB (Diez et al., 2001)
a. Denaturing: 95°C for 5 min
b. 40 cycles: 94°C for 20 s, 54 °C
for 20 s, and 72°C for 80 s.
c. Final extension: 72°C for 10 min.
d. Melting curveb
EukB (Diez et al., 2001)
as in reference
Euk516R (Diez et al., 2001)
as in reference
907R(Cassamayor et al., 2000)
a. Denaturing: 95°C for 5 min
b. 40 cycles: 94°C for 20 s, 56 °C
for 20 s, and 72°C for 60 s.
c. Final extension: 72°C for 7 min.
d. Melting curveb
a
All forward and reverse primers had M13 forward (GTAAAACGACGGCCAGT) and reverse (CAGGAAACAGCTATGACC)
adapters, respectively, on 5' end, which was used as target to sequence the resulting amplicons.
b
Melting curve for all reactions was performed as following: 95°C for 5 s, 52°C for 1 min, and a continuous temperature ramp
(0.11°C/s) from 52 to 97°C.
Table S2. The number of analyzed SAGs and the recovered SSU rRNA gene sequences
Fraction (SAG name)
Putative heterotrophic protists
(AAA071_)
Putative mixotrophic protists
(AAA074_)
SAGs
a18S
b16S
rRNA
rRNA
mitochondria
chloroplasts
prokaryotes
315
47 (15%)
5 (1.5%)
0
12 (4%)
315
38 (12%)
22 (7%)
66 (21%)
17 (5%)
nd
nd
nd
156 (25%)
Bacterioplankton
630
aSAGs yielding 18S rRNA gene sequences
bSAGs yielding 16S rRNA gene sequences
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