Hiseq 1500 platform

D39 and ΔTCS07 were inoculated to an OD₆₀₀ of 0.005 in C+Y in quadruplicates. Cultures were grown to OD₆₀₀ of 0.5, at which point they were harvested. RNA was extracted as described above. 2.5 μg total RNA was depleted for ribosomal RNA with the Ribo-Zero rRNA Removal Kit for Gram-Positive Bacteria (Illumina). Subsequent library preparation was performed using the NEBNext RNA library prep kit for Illumina. Library quality was assessed using the Fragment Analyzer followed by library qPCR quantification (Kapa library quantification kit). Sequencing was carried out on a HiSeq1500 platform (Illumina). Raw-reads were mapped to D39 (assembly accession: GCA_000014375.1) using Bowtie2 [78 (link)] and quantified using the RNA-seq quantification pipeline in SeqMonk (Babraham Bioinformatic). Differential expression was calculated using the Bioconductor package EdgeR in R [79 (link)], and illustrative representation of data was created using the Circlize package [80 (link)]. Data are deposited in the GEO and SRA database with accession numbers GSE132733 and SRP201427, respectively.

Viral RNA was extracted using a QIAamp viral RNA isolation kit and sequenced by the UTMB Sequencing Core. cDNA libraries were prepared using random hexamer priming (TruSeq RNA library prep kit) and sequenced on an Illumina HiSeq 1500 platform. Reads were trimmed of adapter sequences and quality controlled for quality scores greater than 30 (Trimmomatic). The reads were aligned to a reference genome (GenBank accession no. AY640589) using Bowtie2 with paired-end, very sensitive local parameters. PCR duplicates were removed with MarkDuplicates (Picard Tools; Broad Institute, MIT). All samples were downsampled to the lowest mean coverage of the data set, 9,984 reads per base. Shannon’s entropy was calculated using custom R scripts over possible nucleotide variables (A, U, C, G, −) using the method described by Nishijima et al (34 (link)). Variants were called using default settings of the software V-Phaser2 (v2.0) (56 (link)). Any variants that did not pass the strand bias test were removed from further analysis.

For mRNA-seq 1 μg total RNA was incubated with poly-dT beads to isolate polyadenylated RNA, subjected to fragmentation and cDNA synthesis followed by library preparation (TruSeq RNA Sample Prep kit v2) performed according to the manufacturer's (Illumina, San Diego, CA, USA) instructions. ChIP-seq libraries were constructed according to the manufacturer's instructions (Illumina) as described previously.^{57 (link)} Sequencing was carried out on a HiSeq1500 platform (Illumina).

Total tissue RNA was extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany) and was analyzed by an Agilent 2100 Bioanalyzer system (Agilent Biotechnologies, Palo Alto, CA) with the RNA 6000 Nano Labchip Kit. Only samples of high‐quality RNA (RNA Integrity Number ≥7.5) were used in the subsequent construction of RNA‐seq libraries. Ribosome‐minus RNA was fractionated from total RNA samples, and RNA‐seq libraries were constructed orderly by RNA fragmentation, random hexamer‐primed cDNA synthesis, linker ligation, and PCR amplification using a TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA). The purified cDNA libraries were sequenced on the Illumina HiSeq1500 platform (paired‐end, 100‐bp read).
Illumina BCL files were converted to FASTQ format using CASAVA v1.8.2. Initial quality controls were conducted by Trimmomatic version 0.32.43 to trim off adapters and low quality bases (ILLUMINACLIP: adapter.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:20). Qualified reads were sent to CIRCexplorer v 1.1.10 pipeline to identify and quantify circRNAs with default parameters 18. A circRNA was called with the support of at least two unique back‐spliced reads. The total number of reads that spanned back‐spliced junctions was used as an absolute measure of circRNA abundance.

mRNA libraries were prepared from 1 ng RNA from five LPS-treated spleens and five spleens of mock-treated chickens each, using the Sense mRNA Seq Library Kit V2 (Lexogen, Vienna, Austria) according to the manufacturer's instructions. Next-generation sequencing (NGS) was performed on the Illumina HiSeq 1,500 Platform (Illumina, Munich, Germany), producing single-end 100 bp reads (Gene Center, Laboratory for Functional Genome Analysis, LMU Munich, Germany). Read filtering, de novo assembly, and alignment to the galGal4 chicken genome was performed in Galaxy (https://usegalaxy.org/) as described elsewhere (41 (link)). FASTA files containing the aligned data were created. Only genes with a minimum 4-fold expression change between groups and an adjusted p-value of < 0.001 were included in further analysis. The home-made scripts used for detailed analysis workflow are available upon request.

All included patients underwent genetic testing. The sequencing technique depended on each treating centers’ preference. Because RiHHTa includes patients from all around Spain, different genetic techniques have been used in the included patients. These techniques include Next generation sequencing (NGS) technologies, Sanger sequencing and other techniques such Multiplex ligation-dependent probe amplification (MLPA). In patients in which NGS was used, coding exons and intronic boundaries of 9 genes related to HHT disease or HHT-like phenotypes (ACVRL1, BMP10, BMPR1A, BMPR2, ENG, GDF2, RASA1, SMAD1, SMAD4) were captured using a custom probe library (SureSelect Target Enrichment Kit for Illumina paired-end multiplexed sequencing method; Agilent Technologies, Santa Clara, California, USA), and sequenced on the HiSeq 1500 platform following Illumina protocols. To determine the pathogenicity of the included variants in the RiHHTa registry, all mutations were checked again by Health in Code SL (A Coruña, Spain) and labeled according to the American College of Medical Genetics and Genomics (ACMG) guidelines [26 (link)]. Whenever a genetic variant was identified through NGS, Sanger sequencing was the preferred used method for familial evaluation, unless technically not feasible.