16S-Seq of stool sample from rheumatoid arthritis patient RA_19 before gut microbiota reduction and fasting
collected on the ward; frozen at -70C and stored at -20C; Complete homogenization by mortaring in liquid nitrogen; DNA extraction with Quick-DNATM Fecal/Soil Microbe Miniprep Kit (Catalog No. D6010, Zymo Research, Brhl, Germany); quantified using with Qubit dsDNA HS (High-Sensitivity) Assay Kit (Thermo Fisher Scientific); 100ng amplified for 16S-, ITS- or 18S- amplicons (core primer Sequences; 16S Primer fw: ACCGCGGCTGCTGGCAC; 16S Primer rev: AGAGTTTGATCMTGGCTCAG; ITS Primer fw: GTCCCTGCCCTTTGTACAC; ITS Primer rev: CCTGGTTAGTTTCTTTTCCTCCGC; 18S Primer fw: AACCTGGTTGATCCTGCCAGT; 18S Primer rev: CTACGAGCTTTTTAACTGCAACAACTTTAATATACGC; Amplification: preincubation at 98C for 5min, 30 cycles; denaturation: 94C for 30sec, annealing cycle 1-5: 56C; cycle 6-30: 65C for 30sec; elongation 72C for 60sec; final step: 72C for 10min. Purification with Ampure-Beads, normalized via Sequelprep-Plates (Thermo Fisher Scientific); ligated to Illumina compatible adapter sequences with a NEB DNA LibraryPrep-Kit. Sequencing: MiSeq instrument with Illumina MiSeq Reagent Kit v3 600-cycle MS-102-3003; read1 and read2: 300 bases.