Bacteroides xylanisolvens exponential phase thiourocanic acid treated replicate 2
Total RNA quality is determined by estimating the A260/A280 and A260/A230 ratios by nanodrop.   RNA integrity is determined by running an Agilent Bioanalyzer gel, which measures the ratio of the ribosomal peaks.  Samples with RIN values of 5 or greater are recommended for library prep. Using the NEBNext rRNA Depletion Kit (Bacteria) (E7850X), bacterial rRNA is depleted starting from 10-1000ng of total RNA by hybridization of rRNA to complementary DNA oligonucleotides, followed by treatment with RNase H and DNase to remove rRNA duplexed to DNA. Next library prep is completed using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760L). Samples are first fragmented at high heat in the presence of divalent cations prior to 1st strand synthesis using random priming.  2nd strand synthesis incorporates dUTPs into the 2nd strand cDNA. End repair creates blunt phosphorylated ends and an adenine overhang is added to the 3 prime end for efficient adapter ligation. The addition of USER enzyme removes the uracil containing strand. The remaining strand is then amplified in a PCR reaction. Indexed libraries that meet appropriate cut-offs for both quantity and quality are quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the LabChip GX or Agilent Bioanalyzer. Samples with a yield of 0.5 ng/ul are used for sequencing. Sample concentrations are normalized to 2.0 nM and loaded onto an Illumina NovaSeq X flow cell at a concentration that yields 35 million passing filter clusters per sample. Samples are sequenced using 100bp paired-end sequencing on an Illumina NovaSeq X according to Illumina protocols. The 10bp unique dual index is read during additional sequencing reads that automatically follow the completion of read 1.  Data generated during sequencing runs are simultaneously transferred to the Yale Center for Genome Analysis (YCGA) high-performance computing cluster. A positive control (prepared bacteriophage Phi X library) provided by Illumina is spiked into every lane at a concentration of 0.3% to monitor sequencing quality in real time. Signal intensities are converted to individual base calls during a run using the system's Real Time Analysis (RTA) software. Base calls are transferred from the machine's dedicated personal computer to the Yale High Performance Computing cluster via a 1 Gigabit network mount for downstream analysis.  Primary analysis - sample de-multiplexing and alignment to the human genome - is performed using Illumina's CASAVA 1.8.2 software suite. The data are returned to the user if the sample error rate is less than 2% and the distribution of reads per sample in a lane is within reasonable tolerance.  Data is retained on the cluster for at least 6 months, after which it is transferred to a tape backup system.