Procedure:
Jejunum/cecum
dissection
and
preparation
of
contents
Mice
are
euthanized
by
cervical
dislocation
at
the
same
time
each
day.
A
12-cm
long
segment
of
jejunum
starting
5
cm
distally
from
the
ligament
of
Trietz
is
dissected
(cecum
is
included).
Cecum
contents
are
extruded
manually
and
snap
frozen
in
liquid
nitrogen
and
stored
at
-80°C.
Procedure:
Extraction
of
microbial
genomic
DNA
Microbial
genomic
DNA
is
extracted
from
mouse
cecum
contents
using
a
protocol
based
on
Ley
et
al
(2008)
where
approximately
100
mg
of
cecum
contents
is
added
to
a
2-mL
screw-caped
tube
containing
1
g
of
silica/zirconia
beads,
500
µL
of
phenol:chloroform:isoamyl
alcohol,
and
210
µL
of
20%
SDS.
Headspace
is
filled
with
cold
DNA
extraction
buffer
(200
mM
Tris
pH
8.0,
200
mM
NaCl,
20
mM
EDTA).
Bead
tubes
are
attached
to
a
MoBio
vortex
adapter
and
shaken
horizontally
at
high
speed
for
10
min.
The
aqueous
phase
is
washed
three
times
with
phenol:chloroform:isoamyl
alcohol
in
phase
gel
lock
tubes.
Nucleic
acids
are
precipitated
with
1
vol
ammonium
acetate
(7.5
M),
2
vol
isopropanol
and
incubated
at
-20°C
for
at
least
1
h.
Precipiated
nucleic
acids
are
concentrated
by
centrifugation
at
15,000
g
for
15
min
then
dissolved
in
TE
buffer.
RNase
A
(100
U)
digestion
is
performed
for
30
min
at
37°C.
Genomic
DNA
is
precipitated
with
0.1
vol
sodium
acetate
(3
M,
pH
5.5)
and
3
vol
ethanol
and
incubated
at
-20°C
for
at
least
1
h.
DNA
is
concentrated
by
centrigugation
at
15,000
g
for
15
min,
pellets
are
washed
twice
with
70%
ethanol,
air
dried
and
dissolved
in
PCR-grade
water
(mock
extractions
without
cecum
contents
are
used
as
negative
controls).
Procedure:
Preparation
and
pyrosequencing
of
SSU
rRNA
gene
amplicon
libraries
V4
amplicons
(V1-2
amplicons
are
reported
in
Campbell
et
al
(2012)
but
are
not
included
in
the
Chesler5
dataset)
are
generated
in
50
µL
reactions
composed
of
1X
AccuPrime
Pfx
reaction
mix,
300
nM
forward
primer,
300
nM
reverse
primer
mix,
1.5
U
AccuPrime
Pfx
polymerase
and
100
ng
of
genomic
DNA.
For
more
information,
see
Campbell
et
al
(2012).
Thermal
profiles
consist
of
a
denaturation
step
at
95°C
for
2
min,
followed
by
27
amplification
cycles
of
95°C
for
15
s,
55°C
for
30
s,
and
68°C
for
45
s,
followed
by
a
final
extension
at
68°C
for
3
min.
Amplicons
are
visualized
on
agarose
gels
for
quality
and
subsequently
purified
using
Agencourt
AMPure
reagents.
A
final
check
of
amplicon
quality
and
quantity
is
performed
on
an
Agilent
Bioanalyzer
using
DNA
1000
reagents.
Sequencing
is
performed
on
a
454-FLX
instrument
following
manufacturer's
instructions.
Sequences
are
extracted
from
raw
FASTA
files
using
the
Ribosomal
Database
Project
(RDP)
Pipeline
Initial
Process.
Mothur
is
used
to
further
screen
sequences
for
each
SSU
rRNA
gene
region.
See
Campbell
et
al
(2012)
for
more
details.
Representatives
of
each
OTU
are
collected
at
genetic
distances
of
0.03
using
Mothur.
RDP
Naive
Bayesian
rRNA
Classifier
is
used
for
taxonomic
analysis
of
all
sequences.
Data
collected
by
investigator
OTU-based
sequence
analysis
and
relative
abudance
*Supplementary
data
are
available
for
this
project.
See
Chesler5