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| Status |
Public on Jun 13, 2023 |
| Title |
HC b_non-IBD colon |
| Sample type |
RNA |
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| Source name |
non-IBD colon
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| Organism |
Homo sapiens |
| Characteristics |
tissue: colon
gender: female
biopsy: descending colon
age: 61
disease: healthy (non-IBD)
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| Treatment protocol |
Surgical samples were fixed in formalin and paraffin embedded (FFPE) for CosMxTM SMI and tissue staining (IHC IF, ISH).
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| Extracted molecule |
total RNA |
| Extraction protocol |
FFPE tissue sections were prepared for CosMxTM SMI profiling. Briefly, five-micron tissue sections mounted on VWR Superfrost Plus Micro slides (cat# 48311-703) were baked overnight at 60°C, followed by preparation for in-situ hybridization (ISH) on the Leica Bond RXm system by deparaffinization and heat-induced epitope retrieval (HIER) at 100°C for 15 minutes using ER2 epitope retrieval buffer (Leica Biosystems product, Tris/EDTA-based, pH 9.0).
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| Label |
SMI imaging barcodes
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| Label protocol |
After HIER, tissue sections were digested with 3 µg/ml Proteinase K diluted in ACD Protease Plus at 40°C for 30 minutes. Tissue sections were then washed twice with diethyl pyrocarbonate (DEPC)-treated water (DEPC H2O) and incubated in 0.00075% fiducials (Bangs Laboratory) in 2X saline sodium citrate, 0.001% Tween-20 (SSCT solution) for 5 min at room temperature in the dark. Excess fiducials were rinsed from the slides with 1X PBS, then tissue sections were fixed with 10% neutral buffered formalin (NBF) for 5 min at room temperature. Fixed samples were rinsed twice with Tris-glycine buffer (0.1M glycine, 0.1M Tris-base in DEPC H2O) and once with 1X PBS for 5 min each before blocking with 100 mM N-succinimidyl (acetylthio) acetate (NHS-acetate, Thermo Fisher Scientific) in NHS-acetate buffer (0.1M NaP, 0.1% Tween pH 8 in DEPC H2O) for 15 min at room temperature. The sections were then rinsed with 2X saline sodium citrate (SSC) for 5 min and an Adhesive SecureSeal Hybridization Chamber (Grace Bio-Labs) was placed over the tissue.
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| Hybridization protocol |
NanoString® ISH probes were prepared by incubation at 95°C for 2 min and placed on ice, and the ISH probe mix (1nM 980 plex ISH probe, 10nM Attenuation probes, 1nM SMI-0006 custom, 1X Buffer R, 0.1 U/μL SUPERase•In™ [Thermo Fisher Scientific] in DEPC H2O) was pipetted into the hybridization chamber. The chamber was sealed to prevent evaporation, and hybridization was performed at 37°C overnight. Tissue sections were washed twice in 50% formamide (VWR) in 2X SSC at 37°C for 25 min, washed twice with 2X SSC for 2 min at room temperature, and blocked with 100 mM NHS-acetate in the dark for 15 min. In preparation for loading onto the CosMx SMI instrument, a custom-made flow cell was affixed to the slide.
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| Scan protocol |
RNA target readout on the CosMx SMI instrument was performed. Briefly, the assembled flow cell was loaded onto the instrument and Reporter Wash Buffer was flowed to remove air bubbles. A preview scan of the entire flow cell was taken, and 15-25 fields of view (FoVs) were placed on the tissue to match regions of interest identified by H&E staining of an adjacent serial section. RNA readout began by flowing 100 μl of Reporter Pool 1 into the flow cell and incubation for 15 min. Reporter Wash Buffer (1 mL) was flowed to wash unbound reporter probes, and Imaging Buffer was added to the flow cell for imaging. Nine Z-stack images (0.8 μm step size) for each FoV were acquired, and photocleavable linkers on the fluorophores of the reporter probes were released by UV illumination and washed with Strip Wash buffer. The fluidic and imaging procedure was repeated for the 16 reporter pools, and the 16 rounds of reporter hybridization-imaging were repeated multiple times to increase RNA detection sensitivity. After RNA readout, the tissue samples were incubated with a 4-fluorophore-conjugated antibody cocktail against CD298/B2M (488 nm), PanCK (532 nm), CD45 (594 nm), and CD3 (647 nm) proteins and DAPI stain in the CosMxTM SMI instrument for 2 h. After unbound antibodies and DAPI stain were washed with Reporter Wash Buffer, Imaging Buffer was added to the flow cell and nine Z-stack images for the 5 channels (4 antibodies and DAPI) were captured.
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| Description |
HC b
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| Data processing |
Images were segmented to obtain cell boundaries, assign transcripts at the cell-level, and obtain a transcript by cell count matrix. Cells with an average negative control count greater than 0.5 and less than 20 detected features were filtered out. After quality control, a mean of 95,3% of cells across samples and FoVs was retained, corresponding to ~46,160 cells per sample on average.
CosMx_normalized_matrix.txt: Counts of molecules for each gene in each segmented cell.
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| Submission date |
Jun 12, 2023 |
| Last update date |
Feb 27, 2024 |
| Contact name |
Azucena Salas |
| E-mail(s) |
asalas1@recerca.clinic.cat
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| Organization name |
IDIBAPS
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| Department |
Gastroenterology
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| Street address |
Rosselló, 149-153
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| City |
Barcelona |
| ZIP/Postal code |
08036 |
| Country |
Spain |
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| Platform ID |
GPL33484 |
| Series (1) |
| GSE234713 |
IBD CosMx NanoString data from colonic mucosa |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM7473683_HC_b_exprMat_file.csv.gz |
9.2 Mb |
(ftp)(http) |
CSV |
| GSM7473683_HC_b_fov_positions_file.csv.gz |
310 b |
(ftp)(http) |
CSV |
| GSM7473683_HC_b_metadata_file.csv.gz |
2.1 Mb |
(ftp)(http) |
CSV |
| GSM7473683_HC_b_tx_file.csv.gz |
310.1 Mb |
(ftp)(http) |
CSV |
| GSM7473683_HCb.tar.gz |
3.0 Gb |
(ftp)(http) |
TAR |
| GSM7473683_HCb_CellLabels.tar.gz |
19.3 Mb |
(ftp)(http) |
TAR |
| GSM7473683_HCb_CompartmentLabels.tar.gz |
20.4 Mb |
(ftp)(http) |
TAR |
| Processed data are available on Series record |
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