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| Status |
Public on Jul 07, 2024 |
| Title |
MT13 |
| Sample type |
SRA |
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| Source name |
H1299MTSS1
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| Organism |
Homo sapiens |
| Characteristics |
cell type: lung adenocarcinoma
cell line: H1299
reporter gene expression: H1299 MTSS1 overexpressed
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| Treatment protocol |
NCI-H1299 cells were overexpressed with MTSS1 mCherry tagged plasmid (GeneCopoeia, Rockville, MD) using HEK293T stable transfection. The cells were put under hygromycin selection pressure and then sorted using BD FACS Diva8.0.1.
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| Growth protocol |
Human lung adenocarcinoma cell line, NCI-H1299 was otained from American Type Culture Collection (ATCC, Manassas, VA) and grown in DMEM (GE Healthcare Life Sciences, Logan, UT), supplemented with 5% Fetal Bovine Serum (Atlanta biological, Lawrenceville, GA) and 1% GlutaMAXTM (Life Technologies, Carlsbad, CA) at 37ºC humidified 5% CO2 atmosphere incubator.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from cell lines using RNeasy Mini kit (Qiagen, Germantown, MD) according to the manufacturer’s protocol. Optical density values of extracted RNA were measured using NanoDrop (Thermo Scientific) to confirm an A260:A280 ratio above 1.9. RNA integration number (RIN) was measured using BioAnalyzer (Agilent Technologies) RNA 6000 Nano Kit.
The cDNA libraries were prepared using the NEXTflex™ Illumina Rapid Directional RNA-Seq Library Prep Kit (BioO Scientific) as per the manufacturer’s instructions. Briefly, polyA RNA was purified from 200 ng of total RNA using oligo (dT) beads. The extracted mRNA fraction was subjected to fragmentation, reverse transcription, end repair, 3’– end adenylation, and adaptor ligation, followed by PCR amplification and SPRI bead purification (Beckman Coulter). The unique index sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies).
Pooled libraries were diluted to 2 nM in EB buffer (Qiagen) and then denatured using the Illumina protocol. The denatured libraries were diluted to 10 pM by pre-chilled hybridization buffer and loaded onto a TruSeq v2 Rapid flow cell on an Illumina HiSeq 2500 and run for 50 cycles using a single-read recipe according to the manufacturer's instructions.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
MT13
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| Data processing |
De-multiplexed and adapter-trimmed sequencing reads were generated using Illumina bcl2fastq (released version 2.18.0.12) allowing no mismatches in the index read. BBDuk was used to trim/filter low quality sequences using “qtrim=lr trimq=10 maq=10” option. Next, alignment of the filtered reads to the human reference genome (GRCh38) was done using HISAT2 (version 2.1.0) applying --no-mixed and --no-discordant options. Read counts were calculated using HTSeq by supplementing Ensembl gene annotation (GRCh38.78).
Genome_build: GRCh38
Supplementary_files_format_and_content: csv file containing raw read counts for each sample
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| Submission date |
Jul 07, 2021 |
| Last update date |
Jul 07, 2024 |
| Contact name |
Yuka Imamura Kawasawa |
| E-mail(s) |
yui102@psu.edu
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| Organization name |
Penn State University
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| Department |
College of Medicine, Pharmacology
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| Street address |
500 University Dr.
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| City |
Hershey |
| State/province |
PA |
| ZIP/Postal code |
17033 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE179644 |
MTSS1 Suppresses NF-kB Activity in Lung Adenocarcinoma through Repression of RelA/p65 Phosphorylation |
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| Relations |
| BioSample |
SAMN20090809 |
| SRA |
SRX11369160 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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