Globinclear

Total RNA was derived from the whole blood, depleted of globin transcripts using Ambion GLOBINclear, and subsequently processed using the Illumina TruSeq v2 library preparation kit. On average, 40 million paired-end reads of 50 bp were generated per participant using illumina’s Hiseq 2000. Samples were demultiplexed using CASAVA and aligned to the hg19 reference genome using STAR [43 (link)]. Alignments were sorted, read groups were added using picard [44 ], and gene expression was quantified using featureCounts [45 (link)]. We selected participants for which all covariates were available (sex, age, BMI, smoking status, and measured cell counts). Raw count matrices per cohort were used for analysis.

Whole blood was collected from patients in the CTRC directly into PAXgene blood RNA tubes (PreAnalytiX, Hombrechtikon, Switzerland), which were stored at −20 °C. RNA was extracted from PAXgene tubes and globin transcript depletion (GlobinClear, ThermoFisher Scientific, MA, USA) was followed by cDNA library preparation using the Illumina TruSeq Stranded mRNA sample preparation kit (Illumina, CA, USA). Globin transcript depletion, cDNA library preparation and RNA sequencing were performed by Beijing Genomics Institute (Shenzhen, China). The sequencing strategy was 70 million 50bp paired-end reads per sample, and sequencing was performed on Illumina (San Diego, CA) HiSeq-2000 sequencers. FASTQ sequences were transferred to the Center for Infectious Disease Research for analysis.

Whole blood RNAseq was performed as previously described (13 (link)) on 11 IMRAS Cohort 1 RAS vaccinees. Whole blood was collected directly into PAXgene blood RNA tubes (PreAnalytiX, Hombrechtikon, Switzerland) and stored at −20°C. RNA extraction and globin transcript depletion (GlobinClear, ThermoFisher Scientific, MA, USA) were performed prior to cDNA library preparation using the Illumina TruSeq Stranded mRNA sample preparation kit (Illumina, CA, USA). Globin transcript depletion, cDNA library preparation and RNA sequencing were performed by the Beijing Genomics Institute (Shenzhen, China). A total of sixty-six RNAseq samples were sequenced, with a target depth of 30 million reads per sample. Samples from four timepoints (day 0, 35, 119, 140) were sequenced on Illumina (San Diego, CA) Hiseq2000 sequencers using 75 base-pair (bp) paired-end reads. The remaining samples were sequenced on BGI500 sequencers using 100 bp paired-end reads.