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| Status |
Public on Apr 17, 2024 |
| Title |
CS9_Lesion1 |
| Sample type |
SRA |
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| Source name |
Lung
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| Organism |
Mus musculus |
| Characteristics |
tissue: Lung
genotype: C3HeB/FeJ
treatment: Necrotic
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| Treatment protocol |
Lungs were removed and placed in BD Cytofix diluted 1:3 with PBS for 24hr at 4°C. Lungs were then washed two times in PBS and incubated in 30% sucrose for 24 hours at 4°C. Lungs were then embedded in OCT and freezing in a dry ice slurry with 100% ethanol. A CM1950 cryostat (Leica) was used to generate 10μm sections. During the sectioning process, lesions were classified as necrotic or non-necrotic by visual inspection (presence of caseum) and brightfield microscopy (assessing alveolar integrity and presence of necrotic debris).
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| Growth protocol |
C3HeB/FeJ mice were infected with Mtb
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| Extracted molecule |
total RNA |
| Extraction protocol |
The fixed frozen sample slides were baked for 2 hours at 60°C to ensure lung tissue adhered to slides. Following baking, we performed target retrieval for 20 minutes following all recommended settings (MAN-10115-04). RNA targets were exposed using recommended concentration and duration of proteinase K (1ug/ml for 15 min). In situ probe hybridization took place overnight (18 hours) using standard hybridization solution with no custom spike-in (v1.0) Mouse NGS Whole Transcriptome Atlas RNA - lot # MWTA12002). The next day, off target probes were removed using stringent washes as recommended. Finally, morphology markers (SYTO13, B220 – PE, CD3 – CF594, and CD11b eF660) were added. Following antibody staining, slides and collection plate were loaded into GeoMx DSP instrument as recommended (MAN-10152-01). Slides were identified and records created for each. Scan parameters were set for each channel: FITC/525 was utilized for SYTO13 nuclear staining, with exposure time of 50ms. Cy3/568nm was used for Alexa 532 to detect B220. Texas Red/615nm was used for Alexa 594 to detect CD3. Cy5/666nm was used for Cy5 to detect CD11b. All non-nuclear exposures were set for 200ms. Configuration files were obtained from the nanostring website. Syto 13 was used for focus. Slides were then scanned. Multiple ROIs were obtained per lesion, including necrotic core (when applicable), inner lesion, outer lesion border, and full thickness (encompassing inner and outer areas), as assessed by nuclear density and autofluorescence pattern. More fine-grained region determination was not possible due to poor performance of antibody staining. ROIs were then collected.
Following collection, GeoMx samples were removed from the machine and allowed to air dry overnight. The following day samples were placed in an open top thermal cycler at 65C for 10 minutes. Next, 10ul of nuclease free water were added to all samples well and pipetted up and down 5 times. PCR was run according to standard GeoMx protocols available in their quick start guide (MAN-10133-03). Pooling and cleanup were also run according to GeoMx protocols, with no deviations. The pooled library was assessed via Bioanalyzer and demonstrated a clean trace. Samples were loaded on the Illumina NextSeq platform at 1.6pM and sequenced twice using the recommended paired end 2 x 27 read acquisition. Sequenced library included 5% PhiX. Fastq files were assessed by QC metrics prior to further analysis.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Lesion
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| Data processing |
Raw probe counts from 2 sequencing runs were combined at the fastq levels and then converted to .DCC files via Nanostring’s geomxngspipeline function. The DCC files were uploaded to the DSP instrument and automatically associated with individual scans. Sequencing and Probe QC was performed using default parameters (Analysis suite version 2.5.1.145). 4 of 32 original samples were removed for low sequencing saturation. Because we intended to pseudobulk ROIs within the same animal and thus required normalization strategies not available on the DSP analysis suite, we exported two datasets: (1) raw, post-qc probe counts and (2) q3 normalized counts (the recommended normalization approach by Nanostring).
To create a pseudobulked data set, we first aggregated all raw, QC-counts originally exported from the DSP, which created 2 sets of data per animal: aggregated counts from granuloma-associated ROIs and counts from distal, uninvolved regions. Due to the removal of samples for low sequencing saturation (see above) 14/16 potential pseudobulk samples remained. Because CLR-transformed values are more appropriately assessed by Aitchison-distance PCoA (which is the Euclidean distance between CLR-transformed samples), PCoA was used for dimensionality reduction.
To perform GSEA, we first calculated log2fc (using the CLR-transformed values) by directly comparing counts between necrotic and non-necrotic granulomas. We then used these log2fc values to rank genes. Ranked gene lists were supplied to a gsea function in R and results for significant enrichment and associated P values were obtained using the C5 Ontology gene sets from MSigDB.
Assembly: mm10
Supplementary files format and content: necrotic_v_non_necrotic_granuloma_clr_counts_and_log2fc.csv: gene expression count data
Supplementary files format and content: 2024_02_27_qc_read_counts_geomx_gern_gerner.csv: log2 fold change gene expression in necrotic vs non-necrotic lesions
Supplementary files format and content: necrotic_v_non_necrotic_granuloma_GSEA.csv: Gene set enrichment analysis of necrotic vs non-necrotic lesions
Library strategy: Spatial Transcriptomics
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| Submission date |
Apr 17, 2024 |
| Last update date |
Apr 18, 2024 |
| Contact name |
Benjamin Henry Gern |
| E-mail(s) |
benjamin.gern@seattlechildrens.org
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| Phone |
2068843161
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| Organization name |
Seattle Children's Research Institute
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| Department |
Center for Global Infectious Diseases Research
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| Lab |
Gern
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| Street address |
307 Westlake Ave N
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE264267 |
GeoMX WGS of Mtb-infected mouse lesions |
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| Relations |
| BioSample |
SAMN40997869 |
| SRA |
SRX24297447 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8215230_DSP-1001660009882-A-A03.dcc.gz |
78.4 Kb |
(ftp)(http) |
DCC |
SRA Run Selector |
| Raw data are available in SRA |
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