RNA sequencing
LYSED
Fresh PBMCs were collected and lysed in TRIzol (Thermo Fisher, Waltham, MA), and total RNA was isolated and purified with an miRNeasy kit (Qiagen, Hilden, Germany). All blood samples at different timepoints from the same subject were processed together. RNA quality and quantity were estimated using Nanodrop (Thermo Scientific, Wilmington, DE) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Before sequencing analysis, all samples were batched according to their age, sex, race, and immunization status, but assayed blindly. Two reference samples were simultaneously processed with the vaccine study samples in each batch. Stranded cDNA sequencing libraries were generated with a TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA) following the manufacturer’s instructions. Briefly, 500 ng of total RNA was used for mRNA selection. After the reverse transcription to first strand cDNA, strand information was maintained with dUTP during second strand synthesis. A single nucleotide (containing adenine) was added to the dsDNA fragments and the products were ligated to an adapter. The products were then purified and amplified by PCR to create the final cDNA library. The library was qualified with an Agilent Bioanalyzer and quantified with a Qubit 2.0 fluorometer. The cluster generation and pair-end (2 × 75 bp) sequencing were performed on an Illumina HiSeq 3000. Up to 96 barcoded samples were pooled for one single run, which yielded at least 30 M passed filter paired reads per sample. Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. The sequencing reads were adapter and quality trimmed and then aligned to the human genome using STAR software, and read counts determined with HTSeqCount software. Raw read counts were normalized using the DESeq2, LIMMA packages, and R software.