RNA-Seq of Homo sapiens: Nasopharyngeal Metatranscriptome
Total RNA quality was assessed using the Fragment Analyzer 5300, and concentrations were measured with a Qubit FLEX fluorometer. RNA libraries were prepared using either the Illumina TruSeq Stranded Total RNA prep kit (20020599) or the Illumina Stranded Total Library Prep kit (20040529), following manufacturer protocols. rRNA depletion was performed with the Illumina Ribo-Zero Plus rRNA Depletion Kit (20037135), and for Batch 5, the Illumina Ribo-Zero Plus Microbiome rRNA Depletion Kit (20072062) was additionally used. For library generation, 100 ng of RNA was used for the TruSeq protocol, with 15 cycles of indexing PCR. For the Stranded Total Library Prep protocol, 20100 ng of RNA was used, with 15 cycles of indexing PCR for 100 ng input and 17 cycles for 100 ng input. Libraries were quantified and assessed using the Qubit FLEX fluorometer and the Fragment Analyzer 5300. Libraries were normalized to 2 nM based on fragment size and concentration and pooled for sequencing. Sequencing was performed on an Illumina NextSeq 2000 using a P3 200 flow cell with 2101 bp paired-end reads, targeting 40 million reads per sample. Demultiplexing was done with the Illumina DRAGEN FASTQ Generation software. Quality control and adapter trimming were performed using Fastp v0.23.1 with default parameters, followed by quality checks with FastQC v0.11.9 and MultiQC v1.12.