Supplemental Materials
Oil accumulation mechanisms of the oleaginous microalga
Chlorella protothecoides revealed through its genome,
transcriptomes and proteomes
Chunfang Gao1,#,$
Email: n1cfgao@sina.com
Yun Wang2,#
Email: wangyun@genomics.cn
Yue Shen2,#
Email: shenyue@genomics.cn
Dong Yan1
Email: severedong588@163.com
Xi He1
Email: 337979924@qq.com
Junbiao Dai1,*
Email: jbdai@tsinghua.edu.cn
Qingyu Wu1,*
Email: qingyu@tsinghua.edu.cn
1MOE
Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University,
Beijing 100084, China
2BGI-Shenzhen,
Shenzhen 518083, China
#
These authors contributed equally to this work.
$
Current address: Department of Criminal Science and Technology, People's Public
Security University of China, Beijing 100038, China
*Corresponding authors:
Dr. Junbiao Dai, Center for Epigenetics and Chromatin, School of Life Sciences,
Tsinghua University, Beijing 100084, China; Phone: +86-10-62796190; Fax:
+86-10-62796190; E-mail: jbdai@tsinghua.edu.cn.
Dr. Qingyu Wu, School of Life Sciences, Tsinghua University, Beijing 100084, China;
Phone: +86-10-62781825; Fax: +86-10-62781825; E-mail:
qingyu@mail.tsinghua.edu.cn
07
10
Cp
st
Ye
a
1900+1640
1120+1100
945
915
815+785+745
680
610
550
450
375
295
225
Supplemental Figure 1: Pulsed field gel electrophoresis of C. protothecoides
chromosomes.
Electrophoresis conditions were as follows: 1% agarose gel in 0.5× TBE buffer with pulse
ramped from 47 sec to 188 sec for 18 h at 5 V/cm.
2
5
Percentage (%)
4
3
2
1
0
0
10
20
Depth (X)
30
40
Supplemental Figure 2: 17 kmer estimation of genome size.
The genome size of C. protothecoides sp. 0710 was estimated to be 27.6 Mb based on
reads from short insert size library.
3
0.01
apo p
last
cell e
e
nvelocell
extern ndomem
extern
bran cell pape
al enal enca
e sys rt
caps psula
tem
ulatin ting envelo
e
s stru pre
extrg
a tructu ctu
part
extraextracceellular re
cellu llular matrix
lar re regio
g
n
intra intracellu intraion part
cellu
ellula
la lar o ca
ne r
macro inr trorganrg
mole acelluelle palle
cular lar part
mem
comp rt
bra
emmembralex
m ly membne-boum
nono
dopte rane-endedborane pane
-mebm
clo rgan rt
branrin synn
e-bo thasseed lumeelle
unde com n
d org plex
a elle
orga organ
nelle elle organ
enve envenelle
lo e lope
o
rg
o
lu e
rgan aneplle
prote
n
elle m lum
in se
e b men
rine/t
rane
periporganemlle
hreo
la
nine
phos protesinmic sppart
a
p
c
h
c
ribofl prote atase omplee
-DN comp x
riboanvin syin
co p lex
ucleo nthasA
le
prote e com
in complexx
mple
D
viral vesiclex
RNANA poly
caps
polym mera
id
s
eras e pro
e II tr cess virionvirion
ansc ivity part
fa
ri
r
ampintion faccto
e in tor
antib
electr
oxiddaing
carb
on tr
o yd
carb
ansp
rate bindinngt
oxyhlic
orter,
b
in
acid
in
trans
bind
d g
chro
ferrin
m catalyintig
g ele
c atin in
ctron
copopfactor b
d gc
s wit
er ch bindin
hin c
a
ytoch
pe ing
n
rome
c cro
lase
b6/f
drug deamyin
e
comp
ansp ase
lex o electr
tr
on aorter
fp
rrier
enzhyotosyscte
glyco
enzyme acti m II
v
m
gen
deberanzymee inhibaittor
nchin regula or
g en tor
h y zym
ion dbrolase
dine
kinas isomin
e regerasg
ulatoe
lipid ligaser
bindin
meta
lc
g
e luster lyas
mom
le talloc bind e
g
nucle
nuccleular trhaaperoin
oside
n
-triph nucleic acidnbsduceer
osph oside indin
nucaletase rebginding
g
o
u
ti
la
d
nutrie e bin tor
ding
oxidnot reserv
oxyg reductaoir
patteen bind se
pepti rn binding
de
ing
phos
phata perobxinding
se re idas
qu
riboinnolinateprotein bgulatoer
ucleo synth indin
g
p
e
ro
small
sele tein btase A
din
strucstructuprotein signanlium bin
tu l ral co activa transinding
g
c
n
d
strura
ti
o
s
u
n
n
ti
c
g
ctura stitue tuent enz er
l con nt of of ce yme
stitue nucle ll wa
subs
ll
trate strucntut of ribaor pore
-spe ral m som
e
te cific tranolecule
transtrapyrro
s
p
o
rt
transcriptionle bindiner
crip
a v g
trans
ator
n ccotifa
criptitranscritio
tr on in ption facctor
to
trans traannscriptiitiation fa
o
s
c
trans
riptio cripti n reg ctorr
lation
n term on re ulato
facto
inatiopressor
r , nu
n fac r
tr
c
a
le
n
r
s
trans trans ic acid ferato
se
mem latio
bran n rebginding
two-c
e tra ulato
omp
sport r
onen
tran
r
t res tran nsporte
pons spos er
vitame regulaase
actin
tor
b din
men DNA pin
alcfiola
anato
t-bas ackin
g
h
o
a
l
mica meta ed pro ging
l stru bolic
ce
bio cture processss
rm
bio logica fo
adh ation
biosloygical lre
sion
nthe gue
carb tic prolation
on
c
cata
bolicutilizatieoss
proc n
cell c cell ad
omm heseioss
u
n
cell c cenllication
ycle cycle
p
cellrodcess
c
ell div eath
cellu
is
lar co
cell ion
ce
ce
mpo
cellullular cocell wallllprolifekrailling
nent
lar co mpon biog tion
cellu
macro
m lar co mpon ent a enesis
cellu
celluoleculemponenetnt biosgsembly
lar m
b
la
d
io
r com syn isas enesis
acro
pone thetic sembly
mole
pro
nt
cular
cellu
la organ cess
comp
le cellu r hom oizsation
cellux subunlar loce
lar m it org alizatatisis
etab aniz on
cellu
o
lic roation
la
chror respocnellular p
cess
moso se to proce
stim ss
e
estab
cytom
kinestiegregaulus
lishm
tio
c
e
p
n
e
e
ro
stablis t of R
cesn
extern esstablish
al en tablish ment hmentNoA localizdeaths
of c f loc atio
m
ca
aliza alizati n
extrpasulatienngt of plo
ro
ti
cellu stru tein on in on
lar str cture localiz cell
ucture orga ation
iz tio
gam organ
macro
ete g niza
mole
ation
e era
n
cular
immguene snile
tion
comp
ne re ncin
sp o n g
macro maclerox subu
lo
c
mole mole nit org alizati se
cule cule aniza on
main maintemetab localiz tion
o p ation
tena
nce noance lic
ro
f caof locacess
tio
ti
memmemlo
b bran on in n
multic
micro mraene orgeadockcinell
ellula
ta
n
tu
izatiog
r org multbule-babolic pro
n
nega mult anism i-org sed
ss
anism procce
al
e
n gtiave regic
process
u llular meta olic
nege
ativetive reglaution oof rganisbm
p cess
e
regu lation biolog al pro
nitro
s
gen lation of c ical rocess
comp of m ellu proc s
ound etab lar pro ess
lic rocess
orgaorganic orgametabo
ce
noph ethe nelle olic p
osph r me orgaprocesss
ate m tabolic niza s
tion
perip
etab
lasm oxid
o
lic proce
pigm ic spa ation reprocesss
posit
c
e
s
d
e
nt m
ive re
etaborganizuction
gulaptiositive
olic p ation
on o regulapore c
p f ce la tion ofomplepxigmenrotacess
positositivellu
r
b
b
ti
c
ive re regulaomponiologicaiogeneson
gulati tion o ent orgl proc is
f ellu aniz ess
o
primn of mecta
atio
la
a
olicr proce n
reguproteinryc metab
regu
lation omp bolic processs
regu lation
s
pro
lex
gula
o
lation of cre
f
b
logicbiogenceess
of ce ellulartion of io
a
b
l
llular comp iolog proc sis
re co po onent bical quaess
regugulatim
nent ioge lity
o
n
org
ne
o
re lati
regugulatioonn of mfecellularanizatisois
lation of m tabo proc n
of re olecu lic pro ess
spon lar fu cess
se to nctio
resp
s
repro rep timulun
ir
rodu
du
respatory ga
ctio s
ons seocutive pro
s exc cesns
resprespones to abio
h
o
resp nse toe to biotitic stimange
onse chem c sti ulus
resp
to ex ical s mulu
onse
tern
tim s
ribon
o al s ulus
resto
ucleo
po ther o timulu
p te
re nse to srgtianisms
secro
mulu
comspponse to
ondin
ary m lex b
s
etab iogesntress
olic p esis
secre
roce
sexu
ti
o
s
n
a
tr
trans anslatil reprodby cesll
mem onal uctio
bran initia n
e tra tion
vesic
tr nspo
le-m
an ansport
ediatr
rt
ted strpositio
ansp n
ort
Percent of genes
CPRO
CVAR
CSUB
Comparison of GO classification
10
1
35
56
51
0.1
Cellular Component
Molecular Function
Biological Process
Supplemental Figure 3: Comparison of genes among C. protothecoides sp 0710, C. variabilis NC64A and C. subellipsoidea C-169.
Every gene from each genome was annotated and clustered based on GO. Each bar represents the number of genes and different
species is color-coded.
4
0
0
0
Number of genes
100
3559
5623
5152
Supplemental Figure 4: The gene families of four sequenced green algae. C.
protothecoides, C. variabilis NC64A, C. subellipsoidea C-169 and C. reinhardtii were
compared. The venn diagram showed the shared and unique gene families. Numbers of
gene families are indicated in black. CPRO: C. protothecoides, CVAR: C. variabilis NC64A;
CSUB: C. subellipsoidea C-169; CREI: C. reinhardtii.
5
Supplemental Figure 5: The phylogenetic tree of H+/hexose cotransporter homologous
in the seven green algae with genome sequenced (C. protothecoides, C. variabilis NC64,
C. subellipsoidea C-169, C. reinhardtii, V. carteri, M. pusilla CCMP1545 and O. tauri). The
phylogenetic tree was generated by Neighbor-Joining method in MEGA 4, and the
bootstrap was set as 1000. All the homologous were categorized into three classes, and
two of the branches which contained proteins in all of the seven algae were defined as
common proteins. However, the third branch which included the three HUP proteins was
only presented in three Chlorella species and the other related alga C. subellipsoidea
C-169, and this group was defined as the HUP-like protein. This kind of protein may be
restricted to Chlorella.
(The Study Accession URL:
http://purl.org/phylo/treebase/phylows/study/TB2:S15529)
6
Supplemental Figure 6: Sequence comparison of HUP1 and the three HUP-like proteins
(Cpr004256.1, Cpr001753.1 and Cpr003452.1) in C. protothecoides.
The 12 transmembrane helixes in Cpr004256.1, Cpr001753.1 and Cpr003452.1 were
predicted according to the structure of HUP1. The 6 conserved amino acids responsible
for hexose recognition were labeled in red.
7
Fig S6
A H
23 Cuts per lane
Trypsin
LC/MS/MS
………
Digestion
(LTQ Orbitrap Velos )
100μg per sample
SEQUEST
Analysis Program
Up regulated: 205
Down regulated: 293
Compare the PSM and Area
The changes ≥ 1.5-fold
674 Proteins
( PSM ≥ 5)
Protein database of
C. protothecoides
1,931 Proteins
identified
Supplemental Figure 7: The main process in the proteomic study of autotrophic and
heterotrophic C. protothecoides.
After one-dimension protein separation, each lane was cut into 23 stripes, and every of
which was digested in gel by trypsin, separately. Then, every sample was analyzed by
LC/MS/MS (LTQ Orbitrap Velos). In protein identification, the 23 results of each lane
were combined and searched against the protein database of C. protothecoides with the
program of SEQUEST. As a result, 1931 proteins were identified in autotrophic and
heterotrophic cells. PSM and area in protein identification were used for the different
expression analysis. In heterotrophic cells, 205 proteins are up-regulated (both PSM and
area are increased ≥ 1.5 fold) and 293 proteins are down-regulated (both PSM and area
are decreased ≥ 1.5 fold)
8
Supplemental Figure 8: Comparison of transcriptome and proteome in autotrophic and
heterotrophic cell.
ARNA and HRNA: transcriptomic gene expression in autotrophic and heterotrophic cell
respectively (coverage >=50% or rpkm>=10); APROT and HPROT: proteomic gene
expression in autotrophic and heterotrophic cell respectively (PSM>=5).
9
Supplemental Figure 9: The functional classification of the genes differently expressed in comparative transcriptomic analysis of
autotrophic and heterotrophic C. protothecoides.
After mRNA sequencing, the expression levels of them in autotrophic and heterotrophic conditions were compared, and the genes
with log2 (fold-change) >1 and P < 0.01 were defined as significantly changed. As a result, in heterotrophic cells 984 genes were
up-regulated and 1136 genes were down-regulated. All of the genes were classified to 9 clusters according to KEGG metabolism
pathway annotation.
10
Nile Red
Chlorophyll
Merge
Bright-field
Heterotrophic
Autotrophic
A
B
CP
LB
CP
Supplemental Figure 10. The elimination of chloroplast after transition from
autotrophic to heterotrophic growth.
(A) Visualization of chloroplast or lipid body by confocal microscopy in autotrophic
(upper) and heterotrophic (lower) cells. Red, Nile Red fluorescence used to show the
lipid body. Green, chlorophyll autofluorescence pseudocolored in green, indicating
the presence of chloroplast. (B) The ultrastructure of C. protothecoides cultivated in
autotrophic and heterotrophic condition using transmission electron microscopy
(TEM). The autotrophic cell (left) contained a cuplike chloroplast (CP). The
heterotrophic cell (right) contained a big lipid body (LB).
11
Supplemental Figure 11: The heat map of PFAM domain with biased distribution in C.
protothecoides.
The number in C. reinhardtii were used as the reference and indicated in black. Red and
green indicated the higher or lower numbers of PFAM in different algae. CPRO: C.
protothecoides, CVAR: C. variabilis NC64A; CSUB: C. subellipsoidea C-169; CREI: C.
reinhardtii. VCAR: V. carteri; MPUS: M. pusilla CCMP1545; OTAR: O. tauri.
12
Supplemental Table 1: Chlorella protothecoides sp. 0710 17 kmer statistics
Species
K
K_num
K_depth
Genome_size
X
C. protothecoides
17
414,205,607
15
27,613,707
16.70
13
Supplemental Table 2: Statistics of the completeness of the genome based on 248 CEGs
Complete
Group 1
Group 2
Group 3
Group 4
#Prots
%Completeness
-
#Total
Average
%Ortho
225
56
51
58
60
90.73
84.85
91.07
95.08
92.31
-
274
62
64
75
73
1.22
1.11
1.25
1.29
1.22
19.56
8.93
21.57
25.86
21.67
Partial
236
95.16
300
1.27
23.31
Group 1
60
90.91
71
1.18
15
Group 2
53
94.64
73
1.38
28.3
Group 3
59
96.72
77
1.31
27.12
Group 4
64
98.46
79
1.23
23.44
“Prots” indicated number of 248 ultra-conserved CEGs present in genome; “% Completeness” indicates
percentage of 248 ultra-conserved CEGs present; “Total” indicates total number of CEGs present including
putative orthologs; “Average” indicates average number of orthologs per CEG; “%Ortho” indicates
percentage of detected CEGS that have more than 1 ortholog.
14
Supplemental Table 3: Repeats in the genome of Cp0710 with combined approaches.
Type
Trf
Repeatmasker
Proteinmask
De novo
Total
a
Repeat Size a
431,366
544,464
564,023
1,028,311
1,397,655
% of genome
1.8817
2.3750
2.4603
4.4856
6.0967
The overlaps between repeats have been excluded before the calculation .
15
Supplemental Table 4: Results of repeat prediction without TRF.
RepeatMasker
Type
Length
(Bp)
% in
genome
ProteinMasker
Length
(Bp)
Combined a
Denovo
% in
genome
Length
(Bp)
% in
genome
Length
(Bp)
% in
genome
DNA
5,342
0.0233
6,327
0.0276
84,696
0.3695
96,207
0.4197
LINE
4,431
0.0193
96
0.0004
84,183
0.3672
88,710
0.3870
SINE
642
0.0028
0
0.0000
0
0.0000
642
0.0028
LTR
15,007
0.0655
14,494
0.0632
41,173
0.1796
66,527
0.2902
Satellite
851
0.0037
449
0.0020
2,919
0.0127
2,921
0.0127
Simple repeat
140,139
0.6113
139,387
0.6080
153,278
0.6686
197,878
0.8632
Low complexity
379,791
1.6567
330,534
1.4418
373,521
1.6293
399,048
1.7407
Tandem Repeat
0
0
73,073
0.3188
0
0.0000
73,073
0.3188
210
0.0009
0
0.0000
302,440
1.3193
302,650
1.3202
544,464
2.3750
564,023
2.4603
1028,311
4.4856
1,123,650
4.9015
Unknown
Total
a
b
b
The length of repeats was calculated and overlaps have been excluded before.
This refers to the repeats that can’t be classified by RepeatMasker.
16
Supplemental Table 5: General statistics of predicted protein-coding genes
De novo
Total number of genes
Average length of mRNA
Average length of cds
Average number of exon
Average length of exon
Average length of intron
Total number of exon
Total number of intron
Homolog
Augustus
SNAP
5473
2960.31
1413.21
7.30
193.66
245.67
39939
34466
7446
2379.82
1137.91
6.35
179.10
231.99
47307
39861
Glimmer
HMM
20068
1068.94
937.18
3.10
301.90
62.61
62297
42229
CREI
CVAR
CSUB
4302
1553.34
888.11
3.91
227.32
228.85
16807
12505
5166
1948.45
1030.37
4.65
221.67
251.65
24013
18847
4782
1714.04
950.78
4.23
224.60
236.07
20243
15461
GLEAN
6247
2598.03
1292.44
6.28
205.73
247.17
39245
32998
CREI: Chlamydomonas reinhardtii; CVAR: Chlorella variabilis NC64A; CSUB: Coccomyxa subellipsoidea
C-169.
17
Supplemental Table 6: Statistics of functional annotation
Type
Total gene
Annotated
All annotated
Database
TrEMBL
Swissprot
KEGG
InterPro
GO
Gene
7039
5725
4537
3910
4599
3559
5831
Percentage (%)
100
81.33
64.46
55.55
65.34
50.56
82.87
18
Supplemental Table 7: Proteins involved in nitrogen transport and assimilation in green algae a.
Reference proteins b
Transporter
XP_001694496.1
nitrate transporter
XP_001694067.1
nitrite transporter
XP_001701575.1
ammonium transporter
XP_001691580.1
urea active transporter
XP_001694885.1
amino acid transporter
Assimilation
XP_001696697.1
nitrate reductase
XP_001696787.1
nitrite reductase
NP_176922.1 c
urease
NP_173602.1 c
urease accessory protein F
AAD16984.1 c
urease accessory protein UREG
NP_850239.1 c
urease accessory protein D
XP_001692927.1
glutamine synthetase
XP_001693082.1
glutamate synthase
XP_001702270.1
glutamate dehydrogenase
XP_001703658.1
nitrogen regulatory protein PII
Urea Cycle
XP_001690709.1
carbamoyl phosphate synthase,
large subunit
XP_001690929.1
ornithine carbamoyltransferase
XP_001696749.1
argininosuccinate synthase
CAA34615.1
argininosuccinate lyase
NP_192629.1 c
arginase
a Only
MPUS
OTAR
VCAR
CSUB
CVAR
CPRO
XP_003057942.1
XP_003081529.1
EFJ43737.1
EIE23748.1
EFN52690.1
XP_003058319.1
XP_003081525.1
EFJ43209.1
EIE18297.1
EFN58263.1
Cpr000340.1
XP_003063809.1
XP_003084401.1
EFJ40601.1
EIE23179.1
EFN53204.1
Cpr001664.1
XP_003083319.1
EFJ41618.1
EIE20547.1
EFN55634.1
XP_003074291.1
EFJ48238.1
EIE22399.1
EFN60084.1
Cpr002375.1
XP_003058323.1
XP_003081526.1
EFJ43675.1
EIE21865.1
EFN52691.1
Cpr000877.1
XP_003057941.1
XP_003081527.1
EFJ43735.1
EIE21866.1
EFN52613.1
Cpr001933.9
XP_003083318.1
XP_003083320.1
XP_003083317.1
XP_003078095.1
EFN50428.1
XP_003057550.1
XP_003074553.1
EFJ51602.1
EIE23502.1
EFN56917.1
Cpr003038.1
XP_003057676.1
XP_003083015.1
EFJ40691.1
EIE24001.1
EFN59782.1
Cpr004691.1
EFJ45751.1
EIE23148.1
EFN55208.1
Cpr003571.2
EFN50797.1
Cpr004333.1
XP_003062922.1
EFJ41943.1
XP_003058904.1
XP_003080815.1
EFJ40705.1
EIE20839.1
EFN52062.1
Cpr002904.1
XP_003056510.1
XP_003074205.1
EFJ50768.1
EIE22039.1
EFN58131.1
Cpr003449.1
XP_003063309.1
XP_003083741.1
EFJ48533.1
EIE18346.1
EFN55601.1
Cpr002598.3
XP_003055265.1
XP_003082446.1
EFJ46826.1
EIE22869.1
EFN52305.1
Cpr003750.5
one protein with the highest score in BlastP (E-value<1E-5) were listed.
b Most
of the reference proteins used for BlastP were proteins of C. reinhardtii.
proteins of Arabidopsis thaliana were used when they were not found in C. reinhardtii. MPUS: Micromonas
pusilla CCMP1545; OTAR: Ostreococcus tauri; VCAR: Volvox carteri; CSUB: Coccomyxa subellipsoidea C-169;
CVAR: Chlorella variabilis NC64A; CPRO: Chlorella protothecoides sp. 0710.
c The
19
Supplemental Table 8: H+/hexose cotransporters in Chlorella kessleri and their homologs
in green algae (BlastP, E-value<1E-5, Alignment length>30%)
Green algae
Homologs
CKES
P15686.2 Q39524.1 Q39525.1
CPRO
Cpr004256.1 Cpr001753.1 Cpr003452.1 Cpr002964.1
Cpr003677.1 Cpr003720.1 Cpr003252.1 Cpr003700.3
CVAR
EFN53774.1 EFN53666.1 EFN50678.1
EFN60010.1 EFN50549.1 EFN54539.1
EFN53468.1 EFN59043.1 EFN52027.1
CSUB
EIE21809.1 EIE25646.1 EIE22313.1 EIE22314.1 EIE19027.1 EIE27221.1
EIE20603.1 EIE22323.1 EIE25022.1 EIE21778.1 EIE25408.1 EIE22371.1
EIE26813.1 EIE26164.1 EIE25526.1 EIE20660.1 EIE21964.1 EIE20094.1
EIE20926.1
CREI
XP_001693177.1 XP_001701103.1
VCAR
EFJ48518.1 EFJ42942.1
MPUS
XP_003063909.1 XP_003062688.1 XP_003054757.1 XP_003062182.1
XP_003059968.1 XP_003059519.1
OTAR
XP_003082978.1 XP_003078139.1 XP_003077948.1 XP_003080102.1
EFN59533.1
EFN58991.1
Cpr005023.1
EFN55620.1
EFN50679.1
CKES: Chlorella Kessler; CPRO: Chlorella protothecoides sp. 0710; CVAR: Chlorella
variabilis NC64A; CSUB: Coccomyxa subellipsoidea C-169; CREI: Chlamydomonas
reinhardtii; VCAR: Volvox carteri; MPUS: Micromonas pusilla CCMP1545; OTAR:
Ostreococcus tauri.
20
Supplemental Table 9: Transcriptome sequencing data statistics.
Culture
condition
Autotrophy
Heterotrophy
Total raw
reads (M)
43.7
40.9
Total raw
base (G)
3.93
3.68
Total clean
reads (M)
40.2
37.9
21
Total clean
base (G)
3.62
3.41
Reads map to
genome (M)
32.4
30.8
Mapping
ratio (%)
80.7
81.2
Supplemental Methods
Pulse Field Gel Electrophoresis
The Pulse field gel electrophoresis (PFGE) was carried out according to Blanc et al.1,
with slight modifications. The algal cells were harvested from 4 day old cultures by
centrifugation at 5,000×g for 5 minutes. Approximately 0.25 ml cell pellets were
re-suspended in 200 μl deionized water, mixed with 2% low melting point agarose in
100 mM EDTA at 42°C, poured into plug molds (Bio-Rad), and placed at 4°C for about
20-30 minutes to harden the agarose. Once solidified, all the plugs were incubatd with
solution V (500mM EDTA, 10mM Tris, pH7.5) overnight at 37°C. On the next day 400 μl
of 5 mg/ml proteinase K in 500 mM EDTA (pH7.5) was added to the tube and gently
inverted a few times to homogenize the solution. They were then incubated overnight
and washed with deionized water. Subsequent washes were done using 1× TE in the
cold room overnight. Electrophoresis was performed under following condition: 1%
agarose gel in 0.5× TBE buffer with pulse ramped from 47 sec to 188 sec for 18 h at 5
V/cm. Saccharomyces cerevisiae chromosomes were used as the PFGE markers (225 to
1900kb).
Supplemental Refereneces
1.
Blanc, G. et al. The genome of the polar eukaryotic microalga Coccomyxa
subellipsoidea reveals traits of cold adaptation. Genome biology 13, R39
(2012).
22