|
| Status |
Public on Nov 06, 2019 |
| Title |
NHEM-c_without_exosomes_control_rep3 |
| Sample type |
SRA |
| |
|
| Source name |
cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: NHEM-c
exosomes: none
passages: 12
|
| Treatment protocol |
A375 and A549 cell-derived exosomes were purified from cell culture supernatants by differential centrifugation. Briefly, culture medium was collected and centrifuged at 400 x g for 10 min to remove whole cells. The supernatant was then centrifuged at 15,000 x g for 20 min to remove debris. This concentrated material was then ultracentrifuged at 100,000 x g for 90 min to generate the exosome pellet. The pellet was resuspended and washed twice with phosphate buffered saline (PBS). The quantity of the exosomes was determined using a Nanodrop ND-1000 spectrophotometer at 420 nm (Thermo Fisher Scientific, Pittsburgh, PA). NHEM-c cells were seeded in a 6-well plate. 250 ul of A375 or A549 cell-derived exosomes determined with an OD420 reading of 0.05 were added in each well twice (in the morning and in the afternoon) at the next day. Total RNAs from cells were isolated after 16 hours of adding tumor cell-derived exosomes.
|
| Growth protocol |
NHEM-c cells were cultured in melanocyte growth medium M2 with supplement mix (PromoCell, Heidelberg, Germany) in a 5% CO2 incubator at 37C. A375 and A549 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% exosome-depleted fetal bovine serum (FBS) and penicillin (100 U/mL)/streptomycin (100 μg/mL). FBS was depleted of exosomes by ultracentrifugation at 100,000 x g for 18 h at 4C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs from cells were isolated using mirVana total RNA isolation kit (Life Technologies), according to the manufacturer’s guidelines. Total RNA was quantified using Nanodrop ND-1000 (Thermo Fisher Scientific). The integrity of these total RNAs was assessed using Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA).
Libraries were prepared using the TruSeq Stranded mRNA LT Sample Prep Kit- Set A (Cat# RS-122-2101) with poly-A enrichment.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
HH-03-MHEM-C-Control-3_S3_0
|
| Data processing |
Illumina FastQ Generation version 1.0.0 used for basecalling.
Quality control (QC) of the raw sequence data was performed using FastQC (version 0.10.1). The FastQC results indicated that all sequences were of high quality (Q32).
Sequences were directly aligned to the Homo sapiens hg38 reference genome assembly (hg38.fa) using tophat2 (version 2.0.13) [6], generating alignment files in bam format.
Differentially expressed genes were identified for the four pairwise comparisons: A375EXO vs. Control, A549EXO vs. Control, A375EXO vs. A549EXO, A375EXO+A549EXO vs. Control using the tuxedo suite of programs including cuffdiff2 (version 2.2.1).
Genome_build: hg38
Supplementary_files_format_and_content: Tab-delimited cuffnorm normalized FPKM values
|
| |
|
| Submission date |
Jul 21, 2016 |
| Last update date |
Nov 06, 2019 |
| Contact name |
Hongying Hao |
| E-mail(s) |
h0hao001@louisville.edu
|
| Phone |
502-852-8404
|
| Organization name |
University of Louisville
|
| Department |
Surgery
|
| Lab |
Dr. Kelly M McMasters
|
| Street address |
505 South Hancock Street
|
| City |
Louisville |
| State/province |
KY |
| ZIP/Postal code |
40202 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE84683 |
Differential expression analysis of RNA-seq data from melanocytes driven by tumor cell-derived exosomes |
|
| Relations |
| BioSample |
SAMN05426752 |
| SRA |
SRX1968298 |
