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| Status |
Public on Apr 11, 2021 |
| Title |
DSP-1001250001620-A05 (D18_LUL_ROI4_PanCK+) |
| Sample type |
SRA |
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| Source name |
LUL
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| Organism |
Homo sapiens |
| Characteristics |
segment: PanCK+
morphology: Alveolar
assay: WTA
race: Black/AA
ethnicity: not provided
Sex: F
age: 65-70
organ failure: Lung
s/s to death (days): 15
donor: D18
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| Extracted molecule |
total RNA |
| Extraction protocol |
Sample extraction was performed according to NanoString GeoMx manufacturer instructions. Slides were stained with CTA or WTA 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. One heart sample was akso stained with anti-aSMA antibody and one LUL and one trachea slide were stained with Syto13 and RNAScope probe against the SARS-CoV-2 S gene instead. 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, aSMA+ and aSMA- for the heart slide, and COVID+ and COVID- for one LUL and one trachea slide. 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. For more information about DSP protocols please see Merritt et al. Nature Biotech 2020 (doi: 10.1038/s41598-020-63539-x)
Each collection of oligo tags from one well (representing an AOI from the tissue section) was indexed with i7xi5 unique dual indexes using GeoMx SeqCode primers with 18 cycles of PCR. After PCR, indexed AOIs were pooled and purified in two rounds of AMPure XP PCR purification using 1.2x bead:sample ratio. Libraries were combined for target counts of 30 counts/um^2 for CTA samples or 150 counts/um^2 for WTA samples and were sequenced on a NovaSeq 6000 S4 flow cell or NextSeq500 HO 75 cycle kit, loading with 5% PhiX.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
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.
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| Data processing |
Library strategy: GeoMx Seq
FastQ files were compiled and demultiplexed with IlluminaBasecallsToFastq (samples indexed with DSP-100166004764) or bcl2fastq (all others)
Adapter trimming with trim galore, trmGaloreOpts = " --hardtrim5 26 --dont_gzip"
Merge overlapping R1 and R2 with flash2, flash2Opts = "-m 26 -e 26 -f 26 -s 1 -r 27"
Extract UMIs in bowtie2, umiExtractOpts = " --bc-pattern=NNNNNNNNNNNNNN"
Align RTS_IDs (probe barcodes) using bowtie2, bowtie2Opts = " --end-to-end -L 4 --trim5 0 --trim3 0 --norc"
Deduplication using UMI-tools, umiDedupOpts = " --edit-distance-threshold=1“
The resulting DCC files were converted to an expression count matrix. AOIs (2 for CTA, 2 for WTA) were removed from the analysis if sequencing reads totaled less than 10,000. Target counts were generated by taking the geometric mean of all probe counts against the same gene target in each well. Individual CTA probes were dropped if they had counts less than 10% of the target counts (2 CTA) or if they were global Grubb’s outliers at alpha = 0.01 (6 for CTA). These probe screens were not performed on the WTA assay as each gene is sampled by one probe only. As a quality control step, sequencing saturation was calculated by dividing the number of non-unique sequencing reads by the total number of reads for each AOI. AOIs with sequence saturation below 50% were dropped from analysis (2 CTA, 0 WTA).
Normalization: The 75th percentile of the target counts of each probe pool in each AOI were calculated and normalized to the geometric mean of the 75th percentiles across all AOIs to give normalization factors. Target counts were divided by these normalization factors to give normalized expression counts.
Supplementary_files_format_and_content: Digital Count Conversion (DCC) file format outputted from GeoMx NGS Pipeline, contains software versions, scan attributes, GeoMx NGS pipeline parameters and output metrics, Q30 scores, and list of deduplicated counts per RTS_ID (probe barcode). Values represented in the collapsed target counts tab are the geometric mean of the probes for a given, removing any targets flagged as outliers. Analyzed counts represent the upper quartile normalized collapsed counts across the study after removing QC flagged segments.
Genome_Build: N/A, sequencing of synthetic tags and alignment to whitelist of RTS_IDs (probe barcodes) found in the config.ini output from the GeoMx DSP platform, see attached NanoString GeoMx .pkc files
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| Submission date |
Dec 09, 2020 |
| Last update date |
Apr 12, 2021 |
| Contact name |
Samouil Farhi |
| Organization name |
Broad Institute of MIT and Harvard
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| Street address |
415 Main St.
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| City |
Cambridge |
| State/province |
Massachusetts |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE162911 |
A single-cell and spatial atlas of autopsy tissues reveals pathology and cellular targets of SARS-CoV-2 [gene expression levels] |
| GSE163530 |
A single-cell and spatial atlas of autopsy tissues reveals pathology and cellular targets of SARS-CoV-2 |
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| Relations |
| BioSample |
SAMN17037951 |
| SRA |
SRX9662097 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4965637_DSP-1001250001620-A05.dcc.gz |
54.7 Kb |
(ftp)(http) |
DCC |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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