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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 05, 2024 |
| Title |
Neonatal thymus day P0.5 (reference), RNA |
| Sample type |
SRA |
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| Source name |
Neonatal Thymus Organ Culture
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| Organism |
Mus musculus |
| Characteristics |
tissue: Neonatal Thymus Organ Culture
strain: C57BL/6N
treatment: Reference
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| Treatment protocol |
The following cytokines were used: Recombinant murine IL-12 (Cat# 210-12, Peprotech, Rocky Hill, NJ). Recombinant murine IL-18 (R&D Systems, #P70380). Recombinant murine TL1A (Peprotech, #1896-TL-010). All lyophilized cytokines were reconstituted in PBS with 0.1% BSA before storing at -80˚C. The concentration of BSA in all vehicle control conditions corresponded to the highest concentration within the specific experiment. Thymuses from neonates (P0.5) were surgically removed, trimmed of fat and connective tissues placed in D-MEM media on ice. 2x- 3x Thymuses per well (indicated for each experiment) were set in 12-well plates over isopore Membrane filters, PC, 0.8 µM, 13 mm (Merck Millipore, #ATTP01300,) in complete media: D-MEM (GIBCO, #31330-038) supplemented with 20 % FCS (TICO EUROPE (Greiner), #71133), 20 μM L-glutamine, 50 μM 2-mercapto-ethanol, 0,8 % penicillin-streptomycin (Sigma-Aldrich, # P4333), 0.4 mM Na pyruvate (Sigma-Aldrich, #Cat S-8636), 1x non-essential amino acids (Sigma-Aldrich, #M7145). 12-well plates were incubated at 37ºC, 5% CO2 for up to 6 days. Cytokines were used at the following concentrations: TL1A [100 ng/mL], IL-12 [4 ng/mL] and IL-18 [40 ng/mL].
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| Growth protocol |
All mice used in this study were C57BL/6N wild-type. Time-mate pregnant females and adult C57BL/6N were purchased by Janvier. All mouse experiments were conducted according to institutional, national, and European animal regulations. Ghent University’s Ethics Committee approved all experimental animal procedures (EC2020-010, EC2022-110 ). Experiments were conducted in agreement with the European Parliament’s Directive 2010/63/EU and the 22-09-2010 Council on the protection of animals used for scientific purposes.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Samples were split up in 3 groups: (1) non-preprocessed samples, (2) T cell-depleted samples and (3) Thymus epithelial cells (TECs)-enriched sample. We isolated 20 neonatal thymuses per condition were pooled together per condition/treatment: [1] (P0.5), [2] NTOC lobes day 1.5 and [3] NTOC lobes day 3 treated with either vehicleor TL1A+IL-18. For the non-processed sample, single-cell suspensions were obtained as described previously. T cell-depleted sample was obtained by negative beads enrichment using biotinylated antibodies as follows: (1) Biotin-anti-CD4 [1:100], (2) Biotin-anti-CD8α [1:100], (3) Biotin-anti-CD19 [1:200] and (4) Biotin-anti-Ter-119 [1:200]. Samples were incubated for 15 min at RT. After 15 min incubation, the cells were centrifuged at 400g, 4ºC for 5 min to remove the excess of non-bound antibodies. 150µL of MagniSort™ Streptavidin Negative Selection Beads (eBioscience, #MSNB-6002) were added per sample and incubated for 10 min at RT. The samples were placed in a MagniSort™ Magnet (eBioscience, #MAG-4902-10) for 5 min at RT and purified-enriched cells were recovered in clean round bottom polystyrene tubes ready for sorting. Thymus epithelial cells (TECs)-enriched samples were obtained by enzymatic digestion with the following digestion mix: RPMI, 2%FCS, 2mM EDTA (Invitrogen, #15575020), 0.125 mg/mL Liberase™TM (Roche, #5401119001), 1mg/mlDispase II (Roche, #04942078001) and 10ng/mlDNase I (Roche, #10104159001). Thymic lobes were incubated for 30–40min in a 37 °C water bath in the enzymatic solution. After digestion, the remaining pieces were filtered through a 70-μm mesh filter and washed with 5–10ml MACS buffer (1× PBS with 5mM EDTA and 2% FCS). Cells were centrifuged at 400g, 4ºC for 5 min. Lastly, cells were subjected to negative bead enrichment with the following biotinylated antibodies: (1) Biotin-anti-CD4 [1:100], (2) Biotin-anti-CD8β [1:100], (3) Biotin-anti-CD19 [1:200], (4) Biotin-anti-CD11b [1:300], and (4) Biotin-anti-Ter-119 [1:200] and FACS sorted based on CD45- EpCAM+. Sorted cells were finally poled in a (1)20:(2)60:(3)20 ratio. Sequencing was done using the following parameters: Novaseq6000 flowcell 100 cycles kit v1.5 paired reads (28-8-0-91), 100 pM + 1 % PhiX.
Thymic NTOC lobes were processed to single-cell suspension by pressing the organ through a 70 μm cell strainer. NTOC supernatant cells were harvested by repeated pipetting of the NTOC well and filter after the lobes were removed from the well. Samples were spun down at 400g, 4ºC for 7 min. Subsequently, samples were washed once more with 12.5 mL of cold PBS and spin down again at 400g, 4ºC for 7 min. Finally, we added 2mL of ACK lysis buffer (Westburg b.v., #10-548E) to each sample for 2 min at RT. Once incubated, we added 10mL of cold PBS to stop the lysis reaction and samples were centrifuged at 400g, 4ºC during 7 min. If needed, lysis step can be repeated again for better removal of red blood cells. Samples were incubated at 37ºC water bath for 45 min and resuspended with a glass pipette each 15 min. Once incubated, we added 5mL of FACS-EDTA buffer and the cells were transferred to a new 15mL falcon tube through a 70µm cell strainer. Subsequent flow cytometry or FACS preparation of all samples was performed on ice. Antibody staining was performed 30min in the dark. For intracellular staining of transcription factors cells were fixed after extracellular staining using eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set (ThermoFisher, #00-5523-00). For intracellular cytokine measurements, cells were first extracted from each tissue as mentioned above, and incubated with Brefeldin A (Enzo Life Sciences, #BML-G405-0025) and Monensin (Biolegend, #420701) for 4 hours, following extracellular staining and fixation and permeabilisation using BD Cytofix/Cytoperm™ (BD Biosciences, #554714). Fixable Viability Dye eFluor™ 780 (ThermoFisher, #65- 0865-14) was used to exclude dead cells in all flow cytometry and FACS experiments. For counting purposes, we added 10 µL/sample of e123count eBeads (ThermoFisher, #01-1234-42) or samples were counted in a BD FACSVerse™ Cell Analyzer. Cell suspensions were analyzed with a BD FACSymphony™ A5, BD FACSymphony™ A3 or BD LSRFortessa™ or purified using a BD Symphony S6, BD FACSAria II or III.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
PTO018_RNA
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| Data processing |
CITE-Seq samples:The Cell Ranger pipeline (10x Genomics, version 3.1.0) was used to perform sample demultiplexing and to generate FASTQ files for read 1, read 2 and the i7 sample index for the gene expression and cell surface protein libraries.
scRNA-Seq samples:The Cell Ranger pipeline (10x Genomics, version 6.0.0) was used to perform sample demultiplexing and to generate FASTQ files for read 1 and read 2 for the gene expression libraries.
Read 2 of the gene expression libraries was mapped to the mouse reference genome.
Subsequent barcode processing, unique molecular identifiers filtering and gene counting were performed using the Cell Ranger suite.
CITE-seq reads were quantified using the feature-barcoding functionality.
Assembly: Sample dependent, see above
Supplementary files format and content: HDF5 Feature-Barcode Matrix Format files as produced by the Cellranger pipeline of 10X Genomics
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| Submission date |
Sep 27, 2023 |
| Last update date |
Sep 05, 2024 |
| Contact name |
Bruno Verstraeten |
| E-mail(s) |
bruno.verstraeten@ugent.be
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| Organization name |
Ghent University
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| Street address |
/
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| City |
Ghent |
| State/province |
Oost-Vlaanderen |
| ZIP/Postal code |
9000 |
| Country |
Belgium |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE244172 |
TL1A and IL-18 synergy promotes GM-CSF dependent thymic emergency granulopoiesis |
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| Relations |
| BioSample |
SAMN37567323 |
| SRA |
SRX21908291 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7808267_PTO018_raw_feature_bc_matrix.h5 |
40.9 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
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