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| Status |
Public on May 01, 2024 |
| Title |
U2OS, pLVX-NAB2-STAT6, 3 days doxycycline, 3' mRNA-seq, biol rep2 |
| Sample type |
SRA |
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| Source name |
U2OS
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| Organism |
Homo sapiens |
| Characteristics |
cell line: U2OS
cell type: osteosarcoma
genotype: pLVX-NAB2-STAT6
treatment: 3 days 1ug/mL doxycycline
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| Treatment protocol |
Lentiviruses were produced in HEK293T cells by co-transfection 8 ug of pLVX-Tet-On Advanced (ClonTech) and pLVX-NAB2-STAT6-FLAG-Tight-Puro. U2OS were passaged into a 6-well plate at 30-40 % confluence. When the cells reached 60-70% confluence, 2 ml of virus medium per well (cell medium with 4 ul of lentiviral particles per ml and 8 ug/ml polybrene (Thermo Fisher, cat#TR1003G)) was added to replace old medium. 24 hours after induction, the virus medium was removed and replaced with fresh cell culture medium for another 48 hours. After that, cells were selected with 200 ug/ml of Neomycin (Corning, cat#MT30234CR) in fresh medium. Cells were selected in Neomycin for two weeks with fresh media being added 3-4 weeks and cells split 1:3 when reaching 80-90% confluency. Then cells were plated into a 6-well plate at 30-40 % confluence. When the cells reached 60-70% confluence, 2 ml of medium fresh lentivirus media generated using pLVX-NAB2-STAT6-FLAG-Tight-Puro with 8 ug/ml polybrene (Thermo Fisher, cat#TR1003G) per well was added to replace the old medium. 24 hours after induction, the virus medium was removed and replaced with fresh cell culture medium for another 48 hours. After that, cells were selected with 0.5 ug/ml of puromycin in fresh medium (InvivoGen, cat#ant-pr-1). 48 hours after selection with puromycin, cells were disassociated with Trypsin and plated at a low density in a 15 cm dish. Single cells were cultured with 0.5 ug/ml of puromycin for the next 2-3 weeks until colonies appeared. Individual microcolonies were moved to a 96-well plate for clonal expansion. Clones were screened after addition of 1ug/mL Doxycline by verified by Western Blot. ATAC-seq was done without doxycycline treatment and after 1, 2, and 3 days of 1ug/mL doxycycline treatment.
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| Growth protocol |
U2OS cells were cultured and maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% super calf serum and Glutmax.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the Zymo RNA Miniprep kitand then used to make sequencing libraries.
QuantSeq 3' - mRNA Seq Library Prep Kit for Illumina (Lexogen)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
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| Data processing |
Image analysis: Firecrest (Illumina pipeline 1.9, default parameters)
base calling: Bustard (Illumina pipeline 1.9, default parameters)
Quality Control: FastQC
Adapter Trimming: TRIM-GALORE
alignment: STAR
tag density files: deepTools package was used to generate BigWiggle files
gene counts: FeatureCounts (Liao et al., 2014)
Assembly: Genome_build: hg19
Supplementary files format and content: Supplementary_files_format_and_content: strand specific bigWig, compatible with UCSC Genome Browser, were generated using the deepTools package (bamCoverage function, with RPGC [reads per genome coverage] normalization). Tab delimited gene count files were generated with FeatureCounts.
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| Submission date |
Dec 08, 2023 |
| Last update date |
May 01, 2024 |
| Contact name |
Alessandro Gardini |
| E-mail(s) |
agardini@wistar.org
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| Phone |
2158983755
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| Organization name |
The Wistar Institute
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| Lab |
Gardini Lab
|
| Street address |
3601 Spruce St, Room 230
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL30173 |
| Series (2) |
| GSE249702 |
NAB2-STAT6 drives an EGR1-dependent neuroendocrine program in Solitary Fibrous Tumors (RNA-Seq) |
| GSE249703 |
NAB2-STAT6 drives an EGR1-dependent neuroendocrine program in Solitary Fibrous Tumors |
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| Relations |
| BioSample |
SAMN38730605 |
| SRA |
SRX22832271 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7959578_U2OS-pLVX-NAB2-STAT6-C1-3days-Dox-rep2_R1_STAR_aln_to_genome_q10_srt_FOR.bw |
7.0 Mb |
(ftp)(http) |
BW |
| GSM7959578_U2OS-pLVX-NAB2-STAT6-C1-3days-Dox-rep2_R1_STAR_aln_to_genome_q10_srt_REV.bw |
6.7 Mb |
(ftp)(http) |
BW |
| GSM7959578_U2OS-pLVX-NAB2-STAT6-C1-3days-Dox-rep2_R1_gene.txt.gz |
925.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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