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| Status |
Public on Sep 25, 2021 |
| Title |
fetal lungs, ILRA-blockade-3 |
| Sample type |
SRA |
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| Source name |
IL-RA-blockade fetal lungs
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| Organism |
Macaca mulatta |
| Characteristics |
gestation age (days): 131
weight at birth (g): 268.4
fetal sex: Female
tissue: fetal lungs
treatment: IL-RA-blockade
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| Treatment protocol |
The Institutional Animal Care and Use Committee at the University of California, Davis approved all animal procedures. Normally cycling, adult female Rhesus macaques (Macaca mulatta) were time mated. At approximately 130 days of gestation (about 80% of term gestation), the pregnant dams received either 1 ml saline solution or 1mg of LPS (Sigma-Aldrich, St. Louis MO) in 1 ml saline solution by ultrasound guided IA injection. IA administration of LPS or saline was performed in mothers of similar weights and ages with fetus with similar fetal genders and gestational ages. Fetuses were surgically delivered 16, 48 or 120 hrs later by cesarean section. Delivered fetuses were euthanized with pentobarbital and tissues collected. There were no spontaneous deaths or preterm labor in the animals. Some pregnant dams were given, in addition to IA LPS, either the IL-1 receptor antagonist (IL-1RA, Kineret® Sobi, Stockholm, Sweden), or the anti-TNF Ab (Adalimubab HUMIRA® AbbVie Chicago, IL), or both. These inhibitors were administered subcutaneously (100mg or 40mg for IL-1RA and anti-TNF respectively) 3hrs before IA LPS as well as IA (50mg or 40 mg for IL-1RA and anti-TNF respectively) 1hr before IA LPS.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The fetal lung was removed, and left lobe was processed into single cell suspensions as described previously 56. The large airways were removed and approximately ~500mg of airway free tissue was collected in a gentleMACS C-tube (Miltenyi Biotec, Auburn, CA) and 5mL digest buffer was added and placed on a gentleMACS octo dissociator for programmed runs. Following dissociation, the cell suspension was passed through a 100µm filter and washed with PBS. After washing, cells underwent red cell lysis (eBioscience, San Diego, CA) along with another pass through a 40um filter after neutralization.
~50,000 cells per lung were submitted for single cell sequencing at the Cincinnati Children’s Hospital Medical Center DNA Sequencing and Genotyping Core. Approximately ~16,000 cells were loaded into one channel of the Chromium system using the 3 prime v3 single cell reagent kit (10X Genomics, Pleasanton, CA). Following capture and lysis, cDNA was synthesized and amplified as per the manufacturer’s protocol (10X Genomics).
The amplified cDNA was used to construct Illumina sequencing libraries that were each sequenced using an Illumina HiSeq 4000 machine.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Raw sequencing data was processed aligned to the Rhesus macaque reference Mmul_8.0.1 (Ensembl version 91) with Cell Ranger version 3.0.2 (10X Genomics) generating expression count matrix files.
Genome_build: Mmul_8.0.1
Supplementary_files_format_and_content: HDF5 Cell Ranger Filtered Count Matrix
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| Submission date |
Mar 22, 2021 |
| Last update date |
Sep 26, 2021 |
| Contact name |
Nathan Salomonis |
| E-mail(s) |
nathan.salomonis@cchmc.org
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| Organization name |
Cincinnati Children's Hospital
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| Department |
Biomedical Informatics
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| Lab |
Nathan Salomonis
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| Street address |
3333 Burnet Avenue
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| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
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| Platform ID |
GPL27943 |
| Series (1) |
| GSE169390 |
Single-cell RNA-Seq of premature Rhesus macaques lungs with intra-uterine inflammation and therapuetic blockade |
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| Relations |
| BioSample |
SAMN18435206 |
| SRA |
SRX10417554 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5201853_ILRA-505-filtered_feature_bc_matrix.h5 |
15.7 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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