|
| Status |
Public on Nov 15, 2018 |
| Title |
Experiment 2, patient 7 |
| Sample type |
SRA |
| |
|
| Source name |
Highly purified neutrophils
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Neutrophils
disease state: Inactive SJIA
serum il-18: 335
|
| Treatment protocol |
Cells were isolated in a two-step isolation with density-gradient sedimentation followed by magnetic-bead based negative selection with the MACSexpress Human Whole Blood Neutrophil Isolation kit (Miltenyi Biotec, Germany), followed by hypotonic lysis of remaining red blood cells. Neutrophil purity was assessed by flow cytometry (see below) and was typically >98%.
|
| Growth protocol |
Freshly isolated
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from purified neutrophils was extracted using the MagMax -96 Total RNA Isolation Kit (Life Technologies)
Using the SuperScript VILO cDNA Synthesis Kit (Life Technologies), 10 ng of RNA was reverse transcribed to make cDNA. The Ion AmpliSeq Library Kit Plus (Life Technologies) and the Ion AmpliSeq Transcriptome Human Gene Expression Core Panel was used to amplify target genes per manufacturer’s directions. Each amplicon was then barcoded with the Ion Express Barcode Adapter (Life Technologies), and purified through Agencourt AMPure XP Beads (Beckman Coulter) and freshly prepared 70% ethanol. Finally, libraries were analyzed with High Sensitivity NGS Fragment Analysis Kit (AATI). All libraries were peaked around 200 bp
Ampliseq transcriptome using Ion Torrent S5
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent S5 |
| |
|
| Description |
Experiment_2_processed_data_file.txt
|
| Data processing |
Amplified reads were mapped with hg19_AmpliSeq_Transcriptome_ERCC_V1 reference from Ion Community.
After reads were mapped they were converted into reads per kilobase of transcript per million mapped reads (RPKM).
Genome_build: hg19
Supplementary_files_format_and_content: RPKM
|
| |
|
| Submission date |
Nov 14, 2018 |
| Last update date |
Nov 15, 2018 |
| Contact name |
Grant Schulert |
| E-mail(s) |
grant.schulert@cchmc.org
|
| Phone |
5136363894
|
| Organization name |
Cincinnati Children's Hospital Medical Center
|
| Street address |
3333 Burnet Ave
|
| City |
CINCINNATI |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
| |
|
| Platform ID |
GPL23934 |
| Series (1) |
| GSE122552 |
Neutrophil gene expression in systemic juvenile idiopathic arthritis |
|
| Relations |
| BioSample |
SAMN10424610 |
| SRA |
SRX5008739 |
