Syto83

TMA slides were processed following the GeoMx® DSP slide preparation user manual (MAN-10087-04). Before being deparaffinized and hydrated by Leica Biosystems BOND RX, the slides were baked in oven at 60 °C for at least 3 h, after which proteinase K was added to digest the proteins. The slides were incubated with WTA probe mix overnight. On the second day, the slides were washed with buffer and stained with GFAP (Invitrogen, 53-9892-82), CD45 (Biolegend, 121302310), and PanCK (Novus, NBP2-33200AF647), and Syto83 (ThermoFisher, S11364) for 2 h. Regions-of-interest (ROIs) were placed on 20X fluorescent images scanned by GeoMx® DSP. Oligoes from PanCK+ and PanCK- regions were collected separately by UV-cleavage. The oligoes then were uniquely indexed using Illumina’s i5 × i7 dual-indexing system. PCR reactions were purified and libraries were paired-end sequenced (2 × 75) on a NextSeq550 system (Illumina). Fastq files were further process by DND system and raw and Q3 normalized counts of all WTA targets in each ROI were obtained through GeoMx® DSP data analysis software. GeoMx® DSP counts from each ROI were scaled to the 75th percentile of expression. The ROIs were categorized according to tissue and spatial groups for subsequent analysis.

FFPE lung tissues from COVID-19-infected hamsters were prepared following the GeoMx digital spatial profiling (DSP) slide preparation user manual (MAN-10115-05; NanoString Technologies, Seattle, WA). Briefly, slides were baked in an oven at 60 °C for at least 30 min and then deparaffinized and rehydrated using the Leica Biosystems BOND-RX (Wetzlar, Germany). After epitope retrieval, tissues were treated with proteinase K and then tissue slides were removed from the Leica Biosystems BOND-RX platform followed by in situ hybridization with an RNA probe mix (mouse whole transcriptome atlas and COVID-19 spike-in panel) at 37 °C overnight. After incubation, tissue slides were washed and stained with morphology markers CD45-647 at 1:10 (Novus Biologicals, Littleton, CO; NBP2-34527AF647), PanCK-488 at 1:20 (eBioscience, Thermo Fisher Scientific; 53-9003-82), and DNA dye Syto83 at 1:10 (Thermo Fisher Scientific; S11364) for 1 h at room temperature.

Autopsy FFPE tissues from COVID-19 infected patents were processed following the GeoMx DSP slide prep user manual (MAN-10087-04). Autopsy slides were baked in oven at 60 °C for at least 1 h, and then deparaffinized and hydrated by Leica Biosystems BOND RX. Proteinase K was added prior to the incubation of incubated with RNA probe mix (CTA and COVID-19 spike-in panel). After overnight incubation, slides were washed with buffer and stained with CD68-594 (Novus Bio, NBP2-34736AF647), CD45-647 (Novus Bio, NBP2-34527AF647), and PanCK-488 (eBioscience, 53-9003-82) and Syto83 (ThermoFisher, S11364) for 1 h, and loaded to the GeoMx DSP machine to scan 20× fluorescent images. Regions of interest (ROIs) were placed by aligning to the ROIs placed during protein profiling. Oligos were cleaved and collected into 96-well plates. Oligos from each AOI was uniquely indexed using Illumina’s i5 × i7 dual-indexing system. 4 μL of a GeoMx DSP sample was used in the PCR reaction. PCR reactions were purified with two rounds of AMPure XP beads (Beckman Coulter) at 1.2× bead-to-sample ratio. Libraries were paired-end sequenced (2 × 75) on a NextSeq550 up to 400 M total aligned reads. Fastq files were processed by the NanoString DND pipeline to generate count files for each target probe, and saved as DCC files. The NCBI GEO accession number for the DSP experiments is GSE159788.

For nucleoid acid staining, cells were incubated with SYTO 83 (Thermo Fisher; concentration, 0.5 μM) for 15 min, centrifuged (5,000 rpm/5 min), and plated on a microscope slide. Snapshots were taken immediately after sample preparation using a Leica DM6 B microscope with a 100× oil immersion objective. Real-time analyses were performed in liquid medium using a CellASIC ONIX platform and compatible B04A plates (Merck), as described previously (23 (link), 33 (link)). In both cases, early-log-phase (OD₆₀₀ ∼ 0.2 to 0.4) M. smegmatis cultures grown in liquid medium were used. For vancomycin-BODIPY (BODIPY FL vancomycin; Thermo Fisher) staining, cells loaded into the observation chamber were exposed to fresh 7H9-OADC-Tween 80 for 5 h, to 7H9-OADC-Tween 80 supplemented with 0.5 μg/ml vancomycin-BODIPY for 5 h, and then to 7H9-OADC-Tween 80 supplemented with 1 μg/ml vancomycin-BODIPY for 5 h (Fig. 3). All microfluidic experiments were performed under constant pressure (1.5 lb/in²). Images were recorded at 10-min intervals using a Delta Vision Elite inverted microscope equipped with a 100× oil immersion objective and an environmental chamber set to 37°C. Pictures were analyzed using the Fiji and R software packages (R Foundation for Statistical Computing, Austria; http://www.r-project.org), including the ggplot2 package (58 ).

Autopsy tissues from COVID-19 infected patents were processed following the GeoMx DSP slide prep user manual (MAN-10087-04). Autopsy FFPE slides were baked in oven at 60 °C for at least 1 h, and then rehydrated and blocked by Nanostring block buffer for 1 h. CD68-594 (Novus Bio, NBP2-34736AF647), CD45-647 (Novus Bio, NBP2-34527AF647), and PanCK-488 (eBioscience, 53-9003-82) were added on the sections along with the Nanostring protein cocktail for overnight incubation Supplementary Table 11. The slides were washed and stained with Syto83 (ThermoFisher, S11364) on the next day. 20X fluorescent images were scanned after loading the slides to GeoMx machine. Regions of interest (ROIs) in Alveoli were selected in both COVID19 high and COVID19 low regions based on the COVID-19 ISH staining in the serial section. Oligos from antibodies were cleaved and collected into 96-well plates. Then these oligos were hybridized with NanoString barcodes overnight and read with an nCounter machine. Digital accounts of each antibody in each ROI were generated for data analysis.

Autopsy FFPE tissues from COVID-19 infected patents were processed following the GeoMx DSP slide prep user manual (MAN-10087–04). Autopsy slides were baked in oven at 60°C for at least 1 hour, and then deparaffinized and hydrated by Leica Biosystems BOND RX. Proteinase K was added prior to the incubation of incubated with RNA probe mix (CTA and COVID-19 spike-in panel). After overnight incubation, slides were washed with buffer and stained with CD68–594 (Novus Bio, NBP2–34736AF647), CD45–647 (Novus Bio, NBP2-34527AF647), and PanCK-488 (eBioscience, 53-9003-82) and Syto83 (ThermoFisher, S11364) for 1 hour, and loaded to the GeoMx DSP machine to scan 20X fluorescent images. Regions of interest (ROIs) were placed by aligning to the ROIs placed during protein profiling. Oligos were cleaved and collected into 96-well plates. Oligos from each AOI was uniquely indexed using Illumina’s i5 × i7 dual-indexing system. 4 uL of a GeoMx DSP sample was used in the PCR reaction. PCR reactions were purified with two rounds of AMPure XP beads (Beckman Coulter) at 1.2x bead-to-sample ratio. Libraries were paired-end sequenced (2×75) on a NextSeq550 up to 400M total aligned reads. Fastq files were further process by DND system and raw and Q3 normalized counts of all CTA targets in each AOI were obtained through Nanostring Azorius program.

4 FFPE slides containing 8 samples (2 tissues per sample) were processed following the GeoMx® DSP slide prep user manual (MAN-10087-04). Before being deparaffinized and hydrated by Leica Biosystems BOND RX, the slides were baked in oven at 67 °C for at least 3 h. 0.1 ug/ml proteinase K was added to digest the proteins for 15 min before the mouse WTA probe mix was added on the slides for overnight hybridization. On the second day, the slides were washed with buffer and stained with mAldh1l1 (ThermoFisher, 702573) for 2 h, and then secondary anti-rabbit AF594 antibody was added on the tissue for 1 h. After 3 times of washing the slides, CD31 (R&D system, AF3628), and Syto83 (ThermoFisher, S11364) were added on the slides for 2 h at room temperature. Regions of interest (ROIs) were placed on 20× fluorescent images scanned by GeoMx^TM DSP. Oligoes from selected regions were collected through UV-cleavage and transferred to 96-well plate. The oligoes then were uniquely indexed using Illumina’s i5 × i7 dual-indexing system. PCR reactions were purified and libraries were paired-end sequenced (2 × 75) on a Novaseq instrument following the GeoMx® NGS library prep user manual (MAN-10117-05). Fastq files were further process by DND system and raw and Q3 normalized counts of all WTA targets in each AOI were obtained through GeoMx^TM DSP data analysis software.