Total RNA was extracted from cells using the Qiagen RNeasy kit. RNA was DNase treated using the TURBO DNA-free kit. Total RNA was quantified using a Qubit 2.0 Fluorometer and evaluated on an Agilent 2100 Bioanalyzer Nano chip for quality assessment. The DNase-treated total RNA (350ng input) was depleted of ribosomal RNA using probes provided by the NEBNext rRNA depletion module according to the manufacturer's recommendations. The library preparation from the rRNA-depleted RNA was conducted according to NEBNext Ultra II Directional RNA kit guidelines. Briefly, the samples were chemically fragmented in a buffer containing divalent cations by heating to 94°C for 15 minutes. The fragmented RNA was random hexamer primed and reverse transcribed to generate the first strand cDNA. The second strand was synthesized after removing RNA template and incorporating dUTP in place of dTTP. The double-stranded cDNA was then end repaired and adenylated at their 3' ends.  A corresponding T nucleotide on the adaptors was utilized for ligating the adaptor sequences to cDNA. The adaptor loop contains a dUTP that is removed along with all other incorporated U's in the second strand by treatment with USER enzyme (Uracil-Specific Excision Reagent). The degradation of the second strand in this step preserves directional sequencing of the intact first strand thus preserving strand information of the RNA. The adaptor ligated DNA was purified using magnetic beads and PCR amplified using 9 cycles to incorporate a unique barcode and generate the final libraries. The final libraries were purified using magnetic beads to remove any remaining primers and adaptors, validated on a TapeStation D1000 tape, normalized to 4nM and pooled equally. The final pool was loaded onto the NextSeq 500 (PLWH samples – Exp630) or NextSeq 2000 (NL4-3 and NI samples – Exp711) P3 flow cell at 750 pM final concentration and sequenced using 2 x 50bp paired-end chemistry. On average, we generated 39 to 53 million reads pass filter per sample.