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Status |
Public on Apr 11, 2021 |
Title |
D20 | 002 | PanCK+ |
Sample type |
other |
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Source name |
LUL
|
Organism |
Homo sapiens |
Characteristics |
infection: SARS-CoV-2 infected x coordinate: 35530.3125 y coordinate: 28738 aoi surface area: 37244.561637 binding density: 0.11 segment: PanCK+ segment: Alveolar race: White ethnicity: not provided Sex: F age: 65-70 organ failure: Lung, heart s/s to death (days): 1
|
Extracted molecule |
other |
Extraction protocol |
[sample isolation protocol] Autopsies were performed at Brigham and Women's Hospital in a negative pressure isolation room by personnel equipped with powered air-purifying or N95 respirators. Organs were removed from the body en bloc, and subsequently dissected for individual organ examination, including weighing and photographing. Representative samples of lung, trachea, and heart were fixed in 10% formalin, processed and paraffin embedded using standard protocols. 5 µm-thick slides were prepared from the FFPE tissue blocks and transferred to the Broad Institute. Sample extraction was performed according to NanoString GeoMx manufacturer instructions. Slides were stained with antibody panels and a SARS-CoV-2 panel spike-ins, all consisting of RNA-ISH probes conjugated with a UV-photocleavable barcodes; along with Syto13, Anti-PanCK, and Anti-CD45 morphological markers. ROIs were selected based on pathologist annotation of morphology, PanCK signal, and S-gene RNAScope from sequential slides. ROIs were segmented into PanCK+ and PanCK- for LUL samples. Regions of interest within the tissue were illuminated with UV light and oligo barcodes were physically aspirated from the tissue and collected into microtiter plates by the GeoMx® Digital Spatial Profiler (DSP) platform.
|
Label |
n/a
|
Label protocol |
n/a
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Hybridization protocol |
Oligonucleotides were then hybridized to fluorescent barcodes and run through the nCounter MAX analysis system to generate raw digital counts. For more information about DSP protocols please see Merritt et al. Nature Biotech 2020 (doi: 10.1038/s41598-020-63539-x)
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Scan protocol |
Oligonucleotides were then hybridized to fluorescent barcodes and run through the nCounter MAX analysis system to generate raw digital counts. For more information about DSP protocols please see Merritt et al. Nature Biotech 2020 (doi: 10.1038/s41598-020-63539-x)
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Description |
raw data file: 20201004_P1001250001721A_P1001250001721A_03.RCC processed data files: Hs P CellDeath v1.0.pkc Hs P COVID19 v1.0.pkc Hs P ImmuneActivation v1.0.pkc Hs P ImmuneCellProfile v1.1.pkc Hs P ImmuneCellTyping v1.0.pkc Hs P IODrugTarget v1.1.pkc Hs P PI3K AKT v1.0.pkc SubR5 Hs P MAPK v1.0.pkc
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Data processing |
The raw counts were inputted into the DSP for calibration (adjusting for oligo-barcode binding efficiency) and for generation of a protein expression matrix; this matrix was subsequently normalized to internal spike-in positive controls (ERCC) to account for system variation. The calibrated and ERRC-normalized expression matrix was then normalized to the geometric mean of housekeeping genes99 as follows: first, the geometric mean of three housekeeping genes, histone H6, GAPDH, and S6, was calculated for each AOI; next, normalization factors were calculated as the geometric mean in each AOI dividing by the geometric mean of geometric means across all AOIs; finally, raw expression values were divided by the calculated normalization factors to give normalized protein expression levels.
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|
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Submission date |
Dec 18, 2020 |
Last update date |
Apr 11, 2021 |
Contact name |
Samouil Farhi |
Organization name |
Broad Institute of MIT and Harvard
|
Street address |
415 Main St.
|
City |
Cambridge |
State/province |
Massachusetts |
ZIP/Postal code |
02142 |
Country |
USA |
|
|
Platform ID |
GPL29514 |
Series (2) |
GSE163529 |
A single-cell and spatial atlas of autopsy tissues reveals pathology and cellular targets of SARS-CoV-2 [protein levels] |
GSE163530 |
A single-cell and spatial atlas of autopsy tissues reveals pathology and cellular targets of SARS-CoV-2 |
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