title: 16S_V3-V4region_nonIBDcontrol_ilealBiospy_patient_64
design: To determine the composition of metabolically-active bacteria, 16S rRNA partial amplicons were generated by PCR from cDNA generated from RNA isolated from ileal biopsies, using primers targeting the V3-V4 region [Fadrosh DW, Ma B, Gajer P, et al. An improved dual-indexing approach for multiplexed 16S rRNA gene sequencing on the Illumina MiSeq platform. Microbiome. 2014;2:6].   Amplicons were purified with MinElute columns (QIAGEN) and finally eluated in 10 microliters of elution buffer. Amplicons were then supplemented with 15 ?l of Nextera PCR master mix and 5 ?l of each index primer. The indexing PCR reaction was performed in a final volume of 50 ?l and it included an initial polymerase activation step at 72?C for 3 min and a DNA denaturing step at 95?C for 30 sec. This was followed by 12 cycles of denaturing at 95?C for 15 sec, annealing at 65?C for 30 sec, and extension at 72?C for 30 sec. A final extension step to fill in incomplete amplification products was conducted at 72?C for 5 min.   In order to remove low molecular weight materials from libraries, indexing PCR reactions were supplemented with 25 ?l of Agencourt AMPure XP paramagnetic beads (Beckman Coulter), mixed, and then incubated for 5 min at room temperature. Beads were separated from supernatant on a magnetic rack. The supernatant was removed and discarded and the beads washed twice for 1 min in 200 ?l 80% ethanol. Beads were allowed to air-dry under the hood and the DNA was finally eluted in 52.5 ?l of EB buffer. To inspect size and concentration of libraries, libraries were analyzed using Agilent high sensitivity DNA kits.   For sequencing, equimolar amounts of each library were pooled and denatured in 200 mM NaOH for 5 min, mixed with ice-cold HT1 buffer to a final concentration of 20 pM and 5% PhiX control libraries, and finally loaded onto a MiSeq (Illumina) instrument and sequenced using a 600-cycle V3 reagent kit and a workflow that demultiplexes libraries and trims sequencing adapters. 