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| Status |
Public on Oct 22, 2020 |
| Title |
MF8 + STAT6i |
| Sample type |
SRA |
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| Source name |
Skin Tumor
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| Organism |
Homo sapiens |
| Characteristics |
tumor type: mycosis fungoides
treatment: STAT6 inhibitor
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| Treatment protocol |
STAT-6 was inhibited by the STAT-6-specific inhibitor AS151749930 (100nM, Axon Medchem)
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| Growth protocol |
All cultures were performed in complete RPMI-1640 medium (ThermoFisher) containing rhIL-2 (10ng/ml, PeproTech) and IL-7 (5ng/ml, PeproTech).16 Cells were activated by the Dynabeads CD3/CD28 T cell Expander (ThermoFisher, bead-to-cell ratio: 1:1).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Skin biopsies were minced and digested enzymatically (Whole Dissociation Skin Kit, Miltenyi Biotec) for 2 hours at 37°C and further dispersed using the Miltenyi gentleMACS Octo Dissociator. Blood samples were purified for CD4+ cells using EasySep enrichment kit (StemCell Technologies). RNA was isolated from samples obtained from SS (n=4), MF (n=3), and HC (n=3) independent donors on 48 hours of culture with/without STAT-6 inhibitor. Total RNA was isolated using the RNeasy purification Kit (Qiagen), and cDNA was obtained employing the High Capacity Reverse Transcription cDNA synthesis kit (Applied Biosystems). RNA was quantified by a Qubit Fluorometer (Invitrogen) and RNA integrity was checked using an Agilent Bioanalyzer and RNA 6000 Nano Kit (Agilent Technologies).
Libraries were prepared using ultra DNA library prep kits. RNA sequencing analysis was performed on Illumina NextSeq500 using 500-bp paired-end reads by Health Science Sequencing core facility at University of Pittsburgh. Between 10 and 30 million read pairs were generated per sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
The sequenced reads were mapped onto the GrCh38 homo sapiens reference genome, version GrCh38.v81 using CLC Genomics Workbench 12.0 (Qiagen) and gene expression values TPM (Transcripts per million) were calculated using CLC Genomics Workbench 12.0.
Paired-end raw read FASTq files were quality checked using the CLC Genomics Workbench 12.0 per default parameters. Failed reads were removed at this time.
Heatmaps were generated in TreeView3 using values normalized for each donor as follows: library size normalization is automatically performed using the TMM (trimmed mean of M values) method. For each desired comparison, gene differential expression was calculated from regularized log-fold-changes and multiple-testing-corrected P-values. Genes were considered to be differentially expressed when P < 0.05 and log2FC was >0.585 in either direction (which corresponds to FC > 1.5 in either direction). Volcano plots, PCA plots, and Venn diagrams were generated from genes that were initially filtered to exclude those with low overall expression in all samples.
Default parameters can be found at: http://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/1200/index.php?manual=Introduction_CLC_Genomics_Workbench.html
Genome_build: GrCh38
Supplementary_files_format_and_content: Excel files with gene expression data
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| Submission date |
Apr 01, 2020 |
| Last update date |
Oct 22, 2020 |
| Contact name |
Patrizia Fuschiotti |
| E-mail(s) |
paf23@pitt.edu
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| Organization name |
University of Pittsburgh
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| Street address |
200 Lothrop St
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| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15261 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE147911 |
Genome-wide transcriptome analysis of the STAT6-regulated genes in advanced-stage cutaneous T-cell lymphoma [Bulk-seq] |
| GSE147947 |
Genome-wide transcriptome analysis of the STAT6-regulated genes in advanced-stage cutaneous T-cell lymphoma |
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| Relations |
| BioSample |
SAMN14518690 |
| SRA |
SRX8042350 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4448830_MF8_STAT6i_S6_R1_001_GE_01112019.xlsx |
7.9 Mb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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