|
| Status |
Public on Sep 11, 2018 |
| Title |
PAX8_Tfx |
| Sample type |
SRA |
| |
|
| Source name |
Reh cells
|
| Organism |
Homo sapiens |
| Characteristics |
facs sorting: GFP (+)
treatment: Transfection
treatment condition: PAX8, 24 hrs.
|
| Treatment protocol |
Two separate transfections of PAX2, PAX5, PAX8, or empty vector (pRRL-CMV-IRES-hrGFPII) control were performed via electroporation in 400µL OPTI-MEM on 1.5X106 REH cells / treatment (BioRad GenePulser Xcell, Square wave, 210V, 15ms, 2x pulse, .1sec gap). The transfected cells were incubated for 24 hours post transfection in normal growth media (RPMI, 10% FBS). Cells were washed twice in PBS plus 1% FBS, and 4-6x105 GFP (+) cells / treatment were harvested via FACS for RNA. Total RNA was isolated from sorted cells using the Qiagen RNeasy Plus kit. The 2 replicates were then combined equally to create one sample per condition for deep sequencing.
|
| Growth protocol |
Reh cells were grown in 10% FBS/RPMI 1640 (Gibco, 11875-093). Cells were grown at 37°C, under 5% CO2 and were passaged every 3-4 days at confluencies suggested by their supplier. Cell aliquots were cultured for no more than ~20 passages.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
PolyA selected mRNA libraries were prepared from total RNA (~ 1 µg per sample) using the Illumina TruSeq Stranded mRNA Library Prep kit.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Sample_TPM_Matrix.csv
|
| Data processing |
Illumina NextSeq System Suite v2.1.3 was used for basecalling.
Cutadapt v1.12 was used to remove adapter sequences and filter low quality bases (Q20 cuttoff).
Reads were aliged to GRCh38 with HISAT2 v2.0.5. Reads mapping to GRCh38.88 defined exons were counted and per gene expression was reported as transcripts per million (TPM) using Stringtie v1.3.1.
Genome_build: GRCh38
Supplementary_files_format_and_content: CSV file containing individual sample expression (TPM) matrix.
|
| |
|
| Submission date |
Jan 30, 2018 |
| Last update date |
Sep 11, 2018 |
| Contact name |
Marshall S Horwitz |
| E-mail(s) |
horwitz@uw.edu
|
| Organization name |
University of Washington
|
| Department |
Pathology
|
| Lab |
Institute for Stem Cell and Regenerative Medicine
|
| Street address |
850 Republican Street N431
|
| City |
Seattle |
| State/province |
Washington |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE109860 |
Activating PAX Gene Family Paralogs to Complement PAX5 Leukemia Driver Mutations |
|
| Relations |
| BioSample |
SAMN08437785 |
| SRA |
SRX3630404 |
