|
| Status |
Public on Aug 22, 2024 |
| Title |
Astrocytes, siAPOE pool, day 2 |
| Sample type |
SRA |
| |
|
| Source name |
astrocytes
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: astrocytes
|
| Treatment protocol |
Cells were seeded in 6-well plates or T-25 flasks until they reached 50-60% confluence and were transfected with siRNA using Lipofectamine RNAiMax to a final concentration of 5 nM for astrocytes. 24 to 48 hours post-transfection cells were washed in PBS and collected by cell scraping for downstream gene expression analysis or for protein analysis. The siRNA targeting AANCR and APOE are listed in the Resource table.
Cells were grown to a confluence of 50% in T-25 flasks or 6-well plates. The AANCR GapmeR (Qiagen GeneGlobe ID:LG00801555-EDA ) was transfected at 10 nM using Lipofectamine 3000 in astrocytes. Four days post-transfection cells were washed in PBS and collected by cell scraping harvested for downstream analysis.
|
| Growth protocol |
Primary human astrocytes (ScienCell) were seeded on poly-L-lysine-coated (2ug/cm2) T-75 culture flasks with complete astrocyte medium (Sciencell). The medium was changed every three days, until cells were approximately 70% confluent and then changed daily until the cell confluence reached 90% confluence. Astrocytes were passaged when cells reach 90-95% confluency.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
From astrocytes treated with siRNA against AANCR, APOE or non-target siRNA, sequencing libraries were prepared using TruSeq Stranded Total RNA Library Prep Kit (Illumina).
From astrocytes treated with siRNA against AANCR, APOE or non-target siRNA, sequencing libraries were prepared using TruSeq Stranded Total RNA Library Prep Kit (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq X |
| |
|
| Data processing |
Sequencing reads were aligned to the GRCh38 (hg38) human reference sequence using GSNAP (v. 2021-12-17) (Wu and Nacu, 2010) with one mismatch allowed, and soft-clipping on.
Expression was quantified using Cufflinks v.2.2.1 (Trapnell et al 2012).
Assembly: hg38
Supplementary files format and content: bigWig (stranded)
|
| |
|
| Submission date |
Apr 12, 2024 |
| Last update date |
Aug 22, 2024 |
| Contact name |
Vivian Cheung |
| E-mail(s) |
jtburd@umich.edu
|
| Organization name |
University of Michigan
|
| Department |
Pediatrics
|
| Lab |
Room 5258
|
| Street address |
210 Washtenaw Ave.
|
| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109-2216 |
| Country |
USA |
| |
|
| Platform ID |
GPL34281 |
| Series (1) |
| GSE263862 |
The enhancer RNA, AANCR, regulates APOE expression in astrocytes and microglia |
|
| Relations |
| BioSample |
SAMN40945467 |
| SRA |
SRX24235782 |
