flow-sorted fecal bacterial sample from Rag1-/- mouse after induction of T cell transfer colitis -2-d12-g14
Colitis was induced by intravenous transfer of 4x105 CD4+CD45RBhiCD25- FACS-sorted T cells into Rag1-/- recipients. Fecal bacteria were sampled before and during fully established colitis (day 12 or 15). Feces were collected, minced with a scalpel, resuspended in PBS and filtered through a 70µm filter. For bulk bacteria, 1x107 bacterial cells were pelleted by centrifugation (7000g, 5’, 4°C) and incubated for 45’ at 95°C with 300µl of a 10% Chelex solution. After centrifugation (7000g,5’,4°C), the supernatant containing the DNA was frozen at -70°C until further usage. For sorted sub-populations, samples were adjusted to an optical density of 0.035 (dλ700nm = 0.5 cm) with PBS. Two mL of the adjusted solution were centrifuged (3.200 g, 10 min, 4°C) and the pellet was resuspended in 1.5 mL of solution A (0.11 M citric acid, 4.1 mM Tween 20 in bidistilled water) and incubated for 20 min. After another centrifugation step, the pellet was resuspended in solution B (0.68 μM 4′,6-diamidino-2′-phenylindole (DAPI, SIGMA), 400 mM Na2HPO4, pH 7.0) and incubated in the dark for 3 h. Cell sorting was performed with a MoFlo cell sorter (DakoCytomation, USA) equipped with a 488 nm argon laser (400 mW) and a ML-UV laser (333-365 nm, 100 mW). Excitation at 488 nm was used to analyze forward scatter (FSC, 488/10) and the ML-UV (450/65) to measure DAPI-DNA fluorescence. Cell sorting was performed using the most accurate sort mode (single and one drop mode: highest purity 99 %, 2.500 cells per second) using either a two-way- or a four-way-sort option at high speed (12 m s-1). Per sample and gate, 500.000 cells were collected. Sorted cells were centrifuged and DNA isolated with Chelex as described above using 70µl 10% Chelex solution. 16s rDNA templates were amplified by PCR using the DNA yield of 2.5x105 bacterial cells. The PCR included 15 pmol of each forward primer 341F 5’-NNNNNNNNNNTCCTACGGGNGGCWGCAG and reverse primer 785R 5’- NNNNNNNNNNTGACTACHVGGGTATCTAAKCC in 20 µL volume of MyTaq buffer containing 1.5 units MyTaq DNA polymerase (Bioline) and 2 µl of BioStabII PCR Enhancer (Sigma). For each sample, the forward and reverse primers had the same 10-nt barcode sequence. PCRs were carried out for 30 cycles using the following parameters: 2 min 96°C predenaturation; 96°C for 15 s, 50°C for 30 s, 70°C for 90 s. DNA concentration of amplicons of interest was determined by gel electrophoresis. About 20 ng amplicon DNA of each sample were pooled for up to 48 samples carrying different barcodes. If needed, PCRs showing low yields were further amplified for 5 cycles. The amplicon pools were purified with one volume AMPure XP beads (Agencourt) to remove primer dimer and other small mispriming products, followed by an additional purification on MinElute columns (Qiagen). About 100 ng of each purified amplicon pool DNA was used to construct Illumina libraries using the Ovation Rapid DR Multiplex System 1-96 (NuGEN).