Immediately after sorting, cells were pelleted by centrifugation and lysed in 350 μl of RLT Plus lysis buffer (Qiagen) supplemented with 2-mercaptoethanol. Lysates were stored at −80°C until RNA isolation using the AllPrep DNA/RNA Micro kit according to the manufacturer’s protocol (Qiagen). RNA quality and quantity were assessed using TapeStation 4200 High Sensitivity RNA tapes (Agilent), and RNA-seq libraries were prepared from 250 pg of total RNA using SMARTer Stranded Total RNA-seq Kit v2 (Takara Bio). Libraries were pooled using dual indexing and sequenced on a NextSeq 500 instrument (Illumina), 75 cycles, single-end, to an average sequencing depth of 19.55M reads.

FASTQ files were generated using bcl2fastq (Illumina). To enable detection of viral RNA, a custom hybrid genome was prepared by joining FASTA, GFF, and GTF files for GRCh37.87, SARS-CoV-2 (NC_045512.2), and Influenza A/California/07/2009 (GCF_001343785.1), which was the dominant strain of influenza throughout BAL fluid collection at our hospital52. An additional negative strand transcript spanning the entirety of the SARS-CoV-2 genome was then added to the GTF and GFF files to enable detection of SARS-CoV-2 replication. Normalized counts tables later revealed high enrichment of SARS-CoV-2 transcripts in diagnosed COVID-19 patients, and enrichment of IAV genes in patients marked as other viral pneumonia. Of note, since our alveolar macrophage sorting strategy for bulk RNA-seq (Extended Data Fig. 2a,,b)b) only focused on CD206hi cells, our bulk RNA-seq data likely underestimate infection of alveolar macrophages infected with SARS-CoV-2 (Fig. 2d).

To facilitate reproducible analysis, samples were processed using the publicly available nf-core/RNA-seq pipeline version 1.4.2 implemented in Nextflow 19.10.0 using Singularity 3.2.1–1 with the minimal command nextflow run nf-core/rnaseq -r 1.4.2 –singleEnd -profile singularity –reverseStranded --three_prime_clip_r2 35355. Briefly, lane-level reads were trimmed using trimGalore! 0.6.4 and aligned to the hybrid genome described above using STAR 2.6.1d56. Gene-level assignment was then performed using featureCounts 1.6.457. Putative sample swaps were identified first by comparing known patient sex with sex determined by levels of XIST and RPS4Y1 expression, followed by single nucleotide polymorphism analysis with NGSCheckMate version 1.0.0 in FASTQ mode using default settings58. Samples exhibiting unexpected correlation were excluded from analysis.

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