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| Status |
Public on Jun 30, 2022 |
| Title |
Sample_03_MEOX |
| Sample type |
SRA |
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| Source name |
peripheral blood
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Treg cells
tissue: peripheral blood
genotype: MEOX1 overexpression
disease state: healty donor
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| Treatment protocol |
Purified Treg cells were stimulated with dynabeads human T-ACT CD3/CD28 beads:cells ratio 1:2, in 96 U-bottomed wells plates and in the presence of IL-2 cytokine (50 ng/mL each). 24h and 48h after stimulation cells were infected with lentiviral particles harboring a custom human ORF for MEOX1 (Sigma-Aldrich, Mission TRC3) or empty backbone-vectors (CTRL), both expressing GFP (Sigma-Aldrich). Cells were cultured for additional 5 days and then stained with fluorochrome-conjugated monoclonal antibodies (CD3-BUV496, CD25-APCR700, CD4 BV570, CD8-BV786, CD127-PE-Cy5) from commercial vendors. Dead cells were excluded from all analyses using Zombie Aqua (BioLegend). GFP+ cells pregated as CD3+CD8-CD4+Aqua-CD25+CD127- were isolated with a FACSAria cell sorter (BD Biosciences).
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| Growth protocol |
PBMCs were isolated from buffy coats from healthy donors via density-gradient separation and cryopreserved in FBS supplemented with 10% DMSO. T regulatory (Treg) cells were enriched using an EasySep Human CD4+CD127lowCD25+ Regulatory T cell Isolation kit.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation of the FACS-purified GFP+ Treg cells was performed following the manufacturer’s protocol using the Quick-RNA Microprep kit (Zymo Research). RNA quality control was performed with the Agilent 2200 Tape Station system and only RNAs having a RIN >8 were used for library preparation.
Libraries for mRNA sequencing were prepared starting from 10 ng tot RNA for each sample by using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech-Takara)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
Lib_MTU_29
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| Data processing |
After demultiplexing with bcl2fastq v2.20 (Illumina, Inc.), quality control checks on raw sequencing data were performed with FastQC v0.11.8.
Adapters removal and dynamic trimming of low-quality bases were performed using Trimmomatic v0.39 (Bolger et al., 2014) with parameters “ILLUMINACLIP:TruSeq2-SE.fa:2:30:10, LEADING:30, MAXINFO:50:0.7, MINLEN:40”.
Single-end reads were aligned to the UCSC hg38 human genome using STAR v2.7.0f (Dobin et al., 2013) with parameters “--outSAMtype BAM SortedByCoordinate, --outFilterMultimapNmax 20, --outWigType wiggle, --outWigNorm RPM”.
Wiggle files were converted to bigWig files using wigToBigWig v2.8 from UCSC tools.
Genome_build: hg38
Supplementary_files_format_and_content: bigWig files RPM normalized
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| Submission date |
Apr 12, 2021 |
| Last update date |
Jun 30, 2022 |
| Contact name |
Alberto Termanini |
| E-mail(s) |
[email protected]
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| Organization name |
Istituto Clinico Humanitas
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| Lab |
Bioinformatic Unit
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| Street address |
Via Manzoni, 113
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| City |
Rozzano |
| State/province |
MI |
| ZIP/Postal code |
20089 |
| Country |
Italy |
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| Platform ID |
GPL21697 |
| Series (2) |
| GSE171895 |
High-dimensional single cell analysis of T cells infiltrating human intrahepatic cholangiocarcinoma [RNA-Seq] |
| GSE171900 |
Hyperactivated Tregs abundantly infiltrate human intrahepatic cholangiocarcinoma and are controlled by transcription factor MEOX1 |
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| Relations |
| BioSample |
SAMN18715122 |
| SRA |
SRX10579806 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5236553_Sample_03_MEOX.bw |
532.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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