Whole Blood and PBMC RNA-seq Profiling

Total RNA was isolated from whole blood collected in PAXgene Blood RNA Tubes using PAXgene Blood RNA Kit (PreAnalytiX) per manufacturer's instructions, and PBMCs using TRIzol (Thermo Fisher Scientific). Total RNA with high quality (RIN > 8) was used for cDNA library preparation using the TruSeq Stranded mRNA Library Preparation kit for NeoPrep (Illumina). Sequencing was performed on an Illumina HiSeq 3000 System in a 1 × 50 bp single read mode. Sequenced reads were mapped against the human reference genome (GRCh37) using TopHat v2.1.1 (7 (link)). Reads mapped to hemoglobin genes were removed from further analysis. Mapped reads were quantified using HTSeq (8 (link)). All the count data were normalized using TCC (9 (link)) and differentially expressed genes were detected using edgeR (10 (link)). Pathway enrichment analysis was performed using Ingenuity Pathway Analysis (IPA) software (Qiagen), respectively. The original RNAseq data is uploaded and available online (Gene Expression Omnibus: GSE118901).

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Oda H., Beck D.B., Kuehn H.S., Sampaio Moura N., Hoffmann P., Ibarra M., Stoddard J., Tsai W.L., Gutierrez-Cruz G., Gadina M., Rosenzweig S.D., Kastner D.L., Notarangelo L.D, & Aksentijevich I. (2019). Second Case of HOIP Deficiency Expands Clinical Features and Defines Inflammatory Transcriptome Regulated by LUBAC. Frontiers in Immunology, 10, 479.

Publication 2019

PAXgene Blood RNA Tubes PAXgene Blood RNA Kit TRIzol TruSeq Stranded mRNA Library Preparation kit for NeoPrep HiSeq 3000 System Ingenuity Pathway Analysis (IPA) software

Blood Cdna library Gene expression Genes Genome human Hemoglobin Human Library Mrna Trizol

Corresponding Organization : National Human Genome Research Institute

Other organizations : National Institutes of Health Clinical Center, Children's Mercy Hospital, Office of Science, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institute of Allergy and Infectious Diseases

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Variable analysis

independent variables

Whole blood collected in PAXgene Blood RNA Tubes
PBMCs using TRIzol (Thermo Fisher Scientific)

dependent variables

Total RNA with high quality (RIN > 8) used for cDNA library preparation
Sequenced reads mapped against the human reference genome (GRCh37) using TopHat v2.1.1
Reads mapped to hemoglobin genes removed from further analysis
Mapped reads quantified using HTSeq
Count data normalized using TCC
Differentially expressed genes detected using edgeR
Pathway enrichment analysis performed using Ingenuity Pathway Analysis (IPA) software (Qiagen)

control variables

Whole blood collected in PAXgene Blood RNA Tubes
PBMCs using TRIzol (Thermo Fisher Scientific)
Total RNA with high quality (RIN > 8)
Sequencing performed on an Illumina HiSeq 3000 System in a 1 × 50 bp single read mode

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