Hiseq 2500 high output flow cell

Frozen gastrocnemius muscles were pulverized using mortar-pestle. Total RNA from 50-100mg tissue powder was isolated using Trizol reagent. RNA was treated with DNAse and purified using NucleoSpin RNA II columns (Machery Nagel) and integrity was confirmed using an Agilent Bioanalyzer. RNA-sequencing was performed at GATC Biotech (Konstanz, Germany). Illumina Stranded TruSeq mRNA Library preparation kit was used with 1 ug of total RNA for the construction of sequencing libraries, which were loaded onto Illumina HiSeq 2500 High-output flow cell and sequenced in a 1 × 50 bp single read format.

Participants collected approximately 1 g of stool and dissolved it in 5 ml RNALater (Ambion, Inc.) pre-aliquoted into Para-Pak vials (Meridian Biosciences, Inc.). In order to maximize our ability to obtain clean genomics signals and avoid experimental and sample processing confounders, great efforts were taken to ensure that all samples were treated in the same manner. Samples were picked up from daily drop-off locations by study coordinators daily, and washed with PBS the same day or no longer than 3 days upon passage before being frozen at −80°C. 29 of the 60 participants returned a complete set of daily stool samples for the entirety of the requested timeseries. Samples were aggregated through time and submitted together for DNA extraction, library preparation and DNA sequencing at the Genomics Platform at Broad Institute of MIT and Harvard. 16 S rRNA regions were amplified from all samples using a universal V4 primer. PreB, day 3 and day 6 samples were also sequenced using shotgun metagenomics sequencing. 16 S rRNA amplicon sequencing was performed on an Illumina MiSeq using v2 chemistry, and metagenomics sequencing was performed on an Illumina HiSeq. 2500 High Output flowcell using v4 chemistry.

The SureSelect Canine Exome and SureSelect XT library preparation kits (Agilent Inc., Santa Clara, CA, USA) were used to prepare the DNA from the blood of an 8-year-old BMD affected with HS for sequencing. Paired end sequencing was carried out using Illumina HiSeq 2500 High Output Flow Cell (2 × 125 bp). Library preparation and sequencing were performed at Michigan State University Research Technology Support Facility Genomics Core. Resulting sequence data were aligned to the CanFam3.1 reference genome using Bowtie2 [34 (link)]. Variant calling was performed using Freebayes and GATK [35 ,36 (link)].