Globin zero kit

The five female patients in the Rockefeller University (RU) Longitudinal Cohort self collected finger stick blood samples. N=336 RNA-Seq samples (mean n=67 per patient) were available for the analysis comparing microbial and human gene expression (Figure 1). Globin-Zero kit (EpiCentre) and the Illumina Truseq mRNA Stranded Library kit, with 11 to 12 polymerase-chain-reaction (PCR) cycles for 5 to 8 nmol per liter input, and sequencing was performed on a HiSeq2500 system (Illumina) with 150-base paired-end reads. Transcript abundance was quantitated with salmon using an hg38 ensGene gene model.

Patients performed fingerstick collection of three drops of blood at home; the blood was placed into a microtainer tube prefilled with fixative, and the specimens were mailed overnight each week to Rockefeller University. RNA was extracted with the PAXgene RNA kit and was purified in accordance with the manufacturer’s protocols, with the exception of the volume of all washes and elutions being decreased to 25% of the volume recommended by the manufacturer. RNA was assessed with the Agilent BioAnalyzer for quantity and quality. For library preparation, we used the Globin-Zero kit (EpiCentre) and the Illumina Truseq mRNA Stranded Library kit, with 11 to 12 polymerase-chain-reaction (PCR) cycles for 5 to 8 nmol per liter input, and sequencing was performed on a HiSeq2500 system (Illumina) with 150-base paired-end reads. Reads were aligned to the human reference genome (hg19) with Gencode, version 18, as the reference gene annotation with the use of STAR software, version 2.3.1z,^{13 (link)} and were quantified with featureCounts software, version 1.5.0-p2.^{14 (link)} Samples with at least 4 million paired-end reads were retained for analysis.