suspended in ice-cold RPMI 1640 and then
applied to a 40%:80% Percoll gradient (GE
Healthcare). After centrifugation at 800gfor
20 min, the IELs were collected from the Percoll
gradient interface and prepared for RNA iso-
lation and purification.
For LPL isolation, the residual tissue frag-
ments were digested for 1 hour at 37°C in RPMI
1640 containing 0.025 mg/ml of Collagenase IV
(Sigma-Aldrich), 0.05 mg/ml of DNase I (Sigma-
Aldrich), and 0.25 units/ml of Dispase (BD
Biosciences). Cells were filtered through 40-mm
cell strainers and applied to a 40%:80% Percoll
gradient (GE Healthcare). After centrifuga-
tion, the LPL were collected from the gradient
interface and prepared for RNA isolation and
purification.
Sample collection from human patients
Procedures involving human subjects were ap-
proved by the UT Southwestern Medical Center’s
Institutional Review Board (STU 112010-130).
Written informed consent was obtained from
all human participants or legal guardians of
participating minors.
Briefly, stool was collected at the time of co-
lonoscopy through direct aspiration of colonic
contents. A total of 10 ml was collected per
individual. Samples were aliquoted into 1-ml
cryovials and immediately frozen in liquid ni-
trogen. Colonic mucosal tissue samples were
collected at time of colonoscopy by standard
endoscopic tissue sampling technique. A total
of two standard forceps biopsies were obtained
per individual. Immediately after collection,
samples were placed in RNAlaterstabiliza-
tion solution (Invitrogen) and stored at−80°C.
RNA extraction was performed using the
Qiagen RNeasy mini kit on thawed RNA-
laterstabilized tissues following the manu-
facturer’s protocol.
Laser capture microdissection
and RNA purification
Epithelial cells were captured from mouse
small intestines by laser capture microdis-
section using an Arcturus PixCell IIe system
as previously described ( 40 ). Total RNA was
isolated from the captured cells using the
PicoPure RNA Isolation Kit (Thermo Fisher,
KIT0204) following the manufacturer’s pro-
tocol. RNA from intact tissue or cells was iso-
lated with the RNeasy Plus Universal Mini Kit
(Qiagen, 73404) following the manufacturer’s
protocol.
Quantitative real-time PCR (qPCR) analysis
of gene expression
cDNA was synthesized from extracted RNA
using M-MLV Reverse Transcriptase (Thermo
Fisher, 28025021) following the manufac-
turer’s protocol. qPCR was performed on a
QuantStudio 7 Flex Real-Time PCR System
(Applied Biosystems) as previously described
( 41 ). Transcript abundances were normalized
toGapdhor 18Sribosomal RNA transcript
abundance, and relative expression values were
calculated by the comparative Ct (DDCt) meth-
od. Primer sequences are provided in table S3.In situ hybridization (ISH)
Plasmids containing the entire open reading
frame of the mouseSprr2a1gene (NCBI acces-
sion: NM_011468.4) with 252 nucleotides were
used as templates of RNA probes. Digoxigenin
(DIG)–labeled antisense or sense probes were
prepared with the HiScribe T7 Quick High
Yield RNA Synthesis Kit (NEB, E2050S) fol-
lowing the manufacturer’s protocol. The
concentrations of riboprobe were determined
on a Nanodrop spectrophotometer, and the
DIG labeling was verified by dot blot using
peroxidase-conjugated anti-DIG antibody
(Roche, 11207733910).
Paraformaldehyde-fixed paraffin-embedded
ileum tissue sections were washed twice in
xylene followed by rehydration in decreasing
concentrations of ethanol. Tissue sections
were permeabilized with 20mg/ml of protein-
ase K for 10 min and treated with ice-cold 20%
acetic acid for 20 s. The sections were then
prehybridized with 50% formamide, 5X saline
sodium citrate, 1 mg/ml of yeast tRNA (Roche,
10109517001), 0.1 mg/ml of heparin (Sigma-
Aldrich, H3149), 1X Denhardt’s solution (Sigma-
Aldrich, D2532), and 0.1% Tween-20 at 60°C for
2 hours and hybridized with 0.5mg/ml riboprobe
at 60°C for 16 hours. After washing, sections
were incubated with peroxidase-conjugated
anti-DIG antibody (Roche, 11207733910), fol-
lowed by biotin-labeled tyramide (Thermo
Fisher, B40951) for signal amplification. Hy-
bridized probes were detected by ABC-Alkaline
phosphatase (Vector Laboratories, AK-5000)
and NBT/BCIP (Roche, 11681451001). Images
were captured using a Zeiss Axio Imager M1
microscope with Zeiss AxioCam MRm Rev3
and Zeiss EC Plan-Neofluar 10X/0.30 M27
objective lens.Intestinal organoid culture
Organoids were cultured from small intestinal
crypts recovered from 6- to 8-week-old mice,
using a protocol based on previously described
methods ( 33 ). The protocol was carried out as
previously described ( 42 ), except that organoid
cultures were passaged every 4 to 7 days.Immunofluorescence microscopy
For paraffin-embedded sections, mouse intes-
tinal tissues were washed with PBS, fixed in
freshly made 4% paraformaldehyde (PFA) at
room temperature for 6 hours, and then em-
bedded in paraffin by the UT Southwestern
histology core. Sections were washed twice
in xylene followed by rehydration in decreas-
ing concentrations of ethanol. Antigen re-
trieval was performed by boiling in 10 mMsodium citrate for 15 min followed by washing
in PBS.
For frozen sections, mouse intestinal tissues
or organoids were snap-frozen in optimum
cutting temperature (OCT) compound (Fisher,
23-730.571). Ten-micron frozen sections were
cut, fixed in 4% PFA at room temperature for
15 min, and washed in PBS. After washing,
slides were blocked with 10% FBS, 1% bovine
serum albumin (BSA), 1% Trixon X-100 in PBS
for1hourandincubatedwiththefollowing
primary antibodies at 4°C overnight: anti-mouse
SPRR2A at 1:100 dilution, anti-Lysozyme at
1:200 dilution, and FITC-conjugated UEA-1 at
20 mg/ml. Secondary antibodies AlexaFluor
488/647 (Thermo Fisher) were diluted 1:400
and applied to slides for 1 hour at room tem-
perature in the dark in a humidified chamber.
Slides were washed and mounted with DAPI
Fluoromount-G (Southern Biotechnology, 0100-
20). Images were captured using a Zeiss Axio
Imager M1 microscope with Zeiss AxioCam
MRm Rev3 and Zeiss EC Plan-Neofluar 10X/
0.30 M27 objective lens.Fluorescence in situ hybridization (FISH)
Mouse intestinal tissues were cut, directly fixed
in Methacarn fixative solution at room tem-
perature for 4 hours, and then embedded in
paraffin by the UT Southwestern histology core.
Sections were washed twice in xylene fol-
lowed by rehydration in decreasing concen-
trations of ethanol. The universal bacterial
probe (/5Alex594N/GCTGCCTCCCGTAGGAGT/
3AlexF594N/) or the control nonspecific
probe (/5Alex594N/ACTCCTACGGGAGGCAGC/
3AlexF594N/) was diluted to 100 nM in FISH
hybridization buffer (20 mM Tris pH 7.2, 0.9 M
NaCl, 0.1% SDS) and applied to slides overnight
at 56°C in a humidified chamber. Slides were
washed and mounted with DAPI Fluoromount-G
(Southern Biotechnology, 0100-20). Images
were captured using a Discover Echo Revolve
microscope.Immunoblot
For protein extraction from intestinal tissue and
stool samples, samples were added to Lysine
Matrix E tubes (MP Biomedicals, 116914050),
and then 1 ml of 1X SDS loading buffer (2% SDS,
10% glycerol, 100 mM DTT, 0.1% bromophenol
blue, and 50 mM Tris-HCl pH 6.8) was added.
Tubes were then added to a FastPrep-24 5G
Homogenizer. After homogenization, the ly-
sates were boiled at 100°C for 10 min and
then centrifuged for 10 min at 4°C at 16,100g.
The supernatants were then transferred to
clean tubes.
For protein extraction from cultured cells and
intestinal organoids, cells were resuspended
in 100ml RIPA buffer (Thermo Fisher, 89900),
and then 100ml of 2X SDS loading buffer
(4% SDS, 20% glycerol, 200 mM DTT, 0.2%
bromophenol blue, and 100 mM Tris-HClHuet al.,Science 374 , eabe6723 (2021) 5 November 2021 9 of 13
RESEARCH | RESEARCH ARTICLE