Fungal ITS amplicon sequencing, mouse fecal sample CX32d4m17, CX3CR1DTR x CD11c cre +/- diphtheria toxin treated
Fungal ITS1-2 regions were PCR amplified using primers ITS1F CTTGGTCATTTAGAGGAAGTAA and TS2R GCTGCGTTCTTCATCGATGC. ITS1 amplicons were generated with 35 cycles using Invitrogen AccuPrime PCR reagents (Carlsbad, CA). Amplicons were then used in the second PCR reaction, using Illumina Nextera XT v2 (San Diego, CA) barcoded primers to uniquely index each sample and 2x300 paired end sequencing was performed on the Illumina MiSeq (Illumina, CA). DNA amplification PCR: Initial denaturation at 94C for 10 min, followed by 40 cycles of denaturation at 94C for 30 s, annealing at 55C for 30 s, and elongation at 72C for 2 min, followed by an elongation step at 72C for 30 min.
CX32_ITS_d4m17