DOI: 10.5061/dryad.tht76hf38
title: CDR3 sequences of germ-free and conventional chickens

description: Microbial colonisation is paramount to the normal development of the
 immune system, particularly at mucosal sites. However, the relationships
 between the microbiome and the adaptive immune repertoire have mostly been
 explored in rodents and humans. Here, we report a high-throughput
 sequencing analysis of the chicken TCRβ repertoire and the influences of
 microbial colonisation on tissue-resident TCRβ+ cells. The results reveal
 that the microbiome is an important driver of TCRβ diversity in both
 intestinal tissues and the bursa of Fabricius, but not in the spleen. Of
 note, public TCRβ sequences (shared across individuals) make a substantial
 contribution to the repertoire. Additionally, different tissues exhibit
 biases in terms of their V family and J gene usage, and these effects were
 influenced by the gut-associated microbiome. TCRβ clonal expansions were
 identified in both colonised and germ-free birds, but differences between
 the groups were indicative of an influence of the microbiota. Together,
 these findings provide insight into the avian adaptive immune system and
 the influence of the microbiota on the TCRβ repertoire.
description: Tissue samples (spleen, bursa of Fabricius, jejunum, caecum, and
 colon) were derived from an in vivo study carried out at the infectiology
 platform PFIE (INRA, Val de Loire) in accordance with the national and
 international regulations specific to the research facility as part of the
 Development of Immune Function and Avian Gut Health (DIFAGH) consortium.
 One group of PA12 white leghorn chickens (n=5) was reared under germ-free
 conditions, whilst another group (n=5) was reared under conventional
 specific pathogen-free (SPF) conditions. For the germ-free birds, the eggs
 were collected immediately after laying and surface sterilized by
 immersion in 1.5% Divosan Plus VT53 (Johnson Diversey, France) for 5
 minutes at room temperature. Subsequently, these eggs were transferred
 into HEPA-filtered incubator. After 18 days at 37°C, the surface of the
 eggs was sterilized in 1.25% Divosan for 4 min at 37 °C. After hatching,
 the temperature of the isolator was maintained at 37.5 °C for 7 days, then
 reduced by 1 °C per day until reaching a stable temperature of 25 °C.
 Chickens were offered X ray-irradiated starter diet (Special Diets
 Services; Dietex, Argenteuil, France) and sterilized water ad libitum. The
 sterility of chickens was confirmed weekly by incubating fresh faecal
 droppings in 10 mL of sterile brain–heart infusion broth under both
 aerobic and anaerobic conditions, which allows for bacterial, yeast, and
 fungal growth. The absence of non-culturable bacteria in faecal and caecal
 samples of germ-free chickens was confirmed by quantitative PCR of a
 conserved region of the bacterial ribosomal 16S gene. Individuals from
 both groups were culled at day 55 post-hatch, when tissue samples were
 harvested and preserved in RNAlater (Thermo Fisher Scientific) according
 to manufacturer's instructions. Samples were stored at –800C prior to
 processing. Tissue samples were weighed and 15 mg of
 each sample was placed in 600 µl of RLT lysis buffer (Qiagen) together
 with 100 µl of 0.2 mm silica beads (Thistle Scientific), and subjected to
 five cycles of 1-minute homogenisation in a Mini-Beadbeater-24 (BioSpec)
 and 30 seconds of cooling on ice. RNA was extracted using the RNeasy Mini
 kit (Qiagen) following the manufacturer’s protocol. For all samples,
 on-column genomic DNA digestion was performed, using the RNase-Free DNase
 Set (Qiagen) in accordance with the manufacturer’s instructions. The
 resulting RNA was eluted in 40 µl of nuclease-free water. The quality and
 integrity of several samples was tested using an RNA ScreenTape (Agilent
 Technologies) on the 4200 TapeStation (Agilent Technologies). The RNA
 samples were stored at –80 °C until further processing.
 5’RACE-ready cDNA was generated using the SMARTer kit (Takara),
 following the manufacturer’s instructions. Subsequently, for each cDNA
 sample, specific 7-bp barcoded forward primers
 (5’-NNNNNNNGAAAAGATGACCACATCTGGTTC-3’) for the chicken TCRβ were used for
 the 5’RACE PCRs. Universal SMARTer kit reverse primers were used for all
 samples, specific to the common 5’ adapter that was added during 5’RACE
 cDNA synthesis. Briefly, for each 25 µl reaction, 5 µl of Phusion 5X
 Buffer (New England Biolabs), 0.5 µl of 10 mM dNTP, 0.5 µl of 10 µM
 UPA-short primer, 0.5 µl of 2 µM UPA-long and 0.25 µl Phusion Hot Start
 Flex DNA Polymerase (New England Biolabs) were added to 15.25 µl
 nuclease-free water, for a total of 22 µl volume. To this, 0.5 µl of the
 10µM gene-specific 7 bp-barcoded primer and 2.5 µl of cDNA were added. The
 individual 25 µl volume 5’RACE PCR reactions were then carried out in
 96-well plates using the thermocycler program recommended by the 5’RACE
 kit (Takara), with 35 cycles of gene-specific amplification with an
 annealing temperature of 60 °C. After PCR amplification, barcoded samples
 were pooled and subjected to electrophoresis on a 1.4% agarose in
 Tris/Borate/EDTA (TBE) buffer gel containing 1:10,000 SYBR green
 (Sigma-Aldrich) at 120 V for 35 minutes. The bands of the expected lengths
 were gel extracted and purified using the QIAquick Gel Extraction Kit
 (Qiagen). DNA libraries of the pooled barcoded PCR
 samples were generated using the NEBNext Ultra II DNA Library Prep Kit for
 Illumina (New England Biolabs), following the manufacturer’s instructions.
 Briefly, this involved the ligation of library adapters, excision, PCR
 enrichment, and clean-up using AMPure XP beads. The quantity and quality
 of the DNA libraries were analysed using the NEBNext Library Quant Kit for
 Illumina (New England Biolabs) and a D1000 DNA tape (Agilent Technologies)
 on the 4200 TapeStation (Agilent Technologies). The libraries were
 sequenced using an Illumina MiSeq platform at the Department of Biology,
 University of Oxford.
description: This is a *.csv file and can be opened with programs such as
 Microsoft Office Excel. For processing and analysis, we recommend the use
 of a program such as R studio, as was done during the data analysis
 described in the publication.