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| Status |
Public on Aug 03, 2023 |
| Title |
Endometrium_01-10_Epithelium_AS-Pre |
| Sample type |
SRA |
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| Source name |
Endometrium
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| Organism |
Homo sapiens |
| Characteristics |
biospy origin: Epithelium
seurat id: run_count_0172_ASH-21Y0018_ENTIRE-1-10-E1
tissue: Endometrium
treatment stage: AS-Pre
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| Treatment protocol |
BMDSC mobilization was performed using granulocyte-CSF (G-CSF) injection at 10 mcgr/kg. CD133+ cells were collected through peripheral blood apheresis and subsequently isolated. Patients were excluded if they did not reach the minimal requirements for mobilized cells (at least 30 million cells with a purity above 70% and viability >50%), presented an unstable medical condition, or refused a central venous catheter when required. Finally, isolated CD133+ cells were delivered into the spiral arterioles of the patient using a minimal invasive radiology intervention through the left brachial artery, reaching each uterine artery using a microcatheter
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| Extracted molecule |
total RNA |
| Extraction protocol |
A two-stage dissociation protocol separated endometrial biopsies into stromal fibroblasts and epithelial enriched single-cell suspensions. Before dissociation, tissue was rinsed with PBS in a petri dish to remove blood and mucus, and excess PBS was removed after rinsing. The tissue was minced into small pieces and dissociated with 3 ml collagenase mix (Collagenase V, Sigma), RPMI+10% fetal bovine serum, and DNaseI (Sigma) at 37ºC under continuous shaking at 175 rpm for 25 min in a 15 mL Falcon tube in a horizontal position. This primary enzymatic step mostly dissociates stromal fibroblasts into single cells while leaving epithelial glands and lumen mostly undigested. The contents were transferred to a 50 mL tube in RPMI media and filtered with a 100 mm cell strainer. The tissue remaining on the filter was used for the epithelial enrichment cell isolation by resuspending and incubating in 10 ml Trypsin mix (Trypsin -EDTA (0.25%) phenol and DNaseI) for 10 min. The resulting two contents were transferred to 50 mL tubes with 20 mL RPMI, filtered with a 100 mm cell strainer, centrifuged, and then resuspended in 1 mL RPMI. Dead cells were removed using the MACS dead cell removal kit (Miltenyi Biotec). Live-cell suspensions were loaded into a Chromium Next GEM Chip G (10X Genomics) to obtain cDNA from individual cells. Mean cells obtained in stromal and epithelial enriched suspensions were 2.38x106 (5x105-6.4x106) and 1.25x106 (2x105-5.3x106), respectively. Viability of stromal cells was 67.44% (31-87%) and 39.5% (20-80%) for epithelial cells.
Total RNA was extracted using the RNeasy Micro Kit with on-column DNase treatment (Qiagen) following the manufacturer's instructions. RNA was resuspended in 14 μl of RNase-free water, and purity and concentration determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher). For cDNA synthesis, 500-1000 ng of total RNA was reverse transcribed following the manufacturer's instructions(Thermo Fisher). 10x-Genomics v3 libraries were prepared as per the manufacturer's instructions. Libraries were sequenced, aiming at a minimum coverage of 50,000 raw reads per cell, on an Illumina NovaSeq 6000 S2v1.5 300 cycles at a mean 40x coverage (paired-end; read 1: 26 cycles; i7 index: 8 cycles, i5 index: 0 cycles; read 2: 98 cycles)
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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 |
10x Genomics
sc_AS_pre_vs_post.h5seurat
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| Data processing |
Processing of scRNA-seq data was conducted with the CellRanger software suite (version 3.1.0). Raw reads were demultiplexed using the mkfastq wrapper command of bcl2fastq (Illumina). The libraries were mapped using GRCh38-3.0.0 as a reference provided by 10x Genomics. The count gene expression matrices per cell were computed per sample using the counts pipeline (with --expect-cells parameter set to default), which performs the steps of read alignment (with STAR mapping tool), UMI counting, calling of cell barcodes, and filtering of empty droplets based on a simple. Processed samples were merged and saved into H5Seurat objects
Assembly: GRCh38 (Ensembl 93)
Supplementary files format and content: H5Seurat single-cell object with barcode metadata
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| Submission date |
Oct 17, 2022 |
| Last update date |
Aug 03, 2023 |
| Contact name |
Raul Pérez-Moraga |
| E-mail(s) |
rperez@fundacioncarlossimon.com
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| Organization name |
Carlos Simon Foundation, INCLIVA Health Research Institute
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| Street address |
Rda. de Narcís Monturiol, 11, Bloque C
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| City |
Paterna |
| State/province |
Valencia |
| ZIP/Postal code |
46980 |
| Country |
Spain |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE215968 |
Decoding the endometrial niche of Asherman's syndrome at single-cell resolution |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM6651713_run_count_0172_ASH-21Y0018_ENTIRE-1-10-E1.tar.gz |
1.3 Mb |
(ftp)(http) |
TAR |
| GSM6651713_run_raw_count_0172_ASH-21Y0018_ENTIRE-1-10-E1.tar.gz |
32.7 Mb |
(ftp)(http) |
TAR |
| Processed data are available on Series record |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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