Bulk fecal bacterial sample from Rag1-/- mouse after induction of T cell transfer colitis -4-d40-unsorted
Colitis was induced by intravenous transfer of 4x10E5 CD4+CD45RBhiCD25- FACS-sorted T cells into Rag1-/- recipients and fecal bacteria sampled during fully established colitis (day 40). Feces were collected, minced with a scalpel, resuspended in PBS, filtered through a 70ľm filter, centrifuged (3000g, 5 min, 4°C), fixed in 2% PFA for 30 minutes at room temperature, centrifuged again, and kept in 70% EtOH at -20°C until further usage. For comparability with an earlier project (SRP069847 in Zimmermann, Hübschmann et al., Eur J Immunol, 2016), samples were adjusted to an optical density of 0.035 (d?700nm = 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 ?M 4?,6-diamidino-2?-phenylindole (DAPI, SIGMA), 400 mM Na2HPO4, pH 7.0) and incubated in the dark for 3 h. 1x10E7 bacterial cells were pelleted by centrifugation (7000g, 5, 4°C) and incubated for 45 at 95°C with 300ľl of a 10% Chelex solution. After centrifugation (7000g,5,4°C), the supernatant containing the DNA was frozen at -70°C until further usage. 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 ľL volume of MyTaq buffer containing 1.5 units MyTaq DNA polymerase (Bioline) and 2 ľl 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).