16S seq human stool - pediatric Crohn's Disease patients
Stool samples were collected and frozen on site at -20C and then shipped to a central laboratory and stored at -80C until further processing. DNA was extracted from ~200 mg stool using the QIAGEN DNeasy PowerSoil HTP 96 Kit (Cat #12955-4) following the manufacturers instructions, including a 2 x 10 minute bead-beating step using the Retsch 96 Well Plate Shaker at speed 20. The V4 region of the 16S rRNA gene was amplified using forward primer 515F (5-GTGCCAGCAGCCGCGGTAA-3) with an error-correcting barcode and 806R (5-GGACTACCAGGGTATCTAAT-3) (13). PCR products were then purified, pooled in equimolar concentrations, and sequenced on three 2 x 300 Illumina MiSeq paired-end runs using reagent kit v3.
SUB4495916
ROT_3_Week0