|
| Status |
Public on Jul 27, 2020 |
| Title |
FXRa1_DMSO_R3 |
| Sample type |
SRA |
| |
|
| Source name |
Cultured cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: HepG2
transgene: FXRa1
selection: Puromycin
treatment: DMSO
treatment duration: 8h
|
| Treatment protocol |
Cells were seeded in 6 well plates and treated for 8 hours with 5 µM Obeticholic acid in DMSO, or DMSO alone (0.1% vol/vol)
|
| Growth protocol |
HepG2 cells were cultured in DMEM 1g/l glucose + 10% FCS in 10cm petri dishes. FXR isoforms or GFP was lentivirally transduced on a pLV(EF1a) IRES-PuroR vector. 2ug/ml puromycin was used for selection of positive clones.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was purified from treated HepG2 using TRIzoL (Invitrogen) reagent according to manufacturer’s instructions. RNA was stored at -80C until further processing
mRNA was isolated using the Poly(A) Beads (NEXTflex). Sequencing libraries were prepared using the Rapid Directional RNA-Seq Kit (NEXTflex)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Replicate 3
FXRa1_DMSO.bw
genes.count_table
|
| Data processing |
Sequencing reads were aligned to the reference genome (hg19 assembly, NCBI37) using Tophat2.18 (--b2-sensitive --microexon-search -p 2 -G Homo_sapiens.GRCh37.87.gtf)
Duplicated reads were removed with Samtools rmdup (v1.9 , using htslib 1.9)
The normalized read numbers per gene were calculated using the cuffnorm feature of Cufflinks (v2.2.1 linked against Boost version 105500) against the Homo_sapiens.GRCh37.87 gene annotation
For visualization, a single file was generated per condition with Samtools merge on the sequenced replicates
Genomic coverage files were generated with deepTools2 bamCoverage (--binSize 1 --normalizeUsing RPGC --effectiveGenomeSize 2736124973) per condition
Genome_build: hg19
Supplementary_files_format_and_content: *.bw BigWig format containing genomic coverage per condition.
Supplementary_files_format_and_content: *.count_table Normalized count values per gene
|
| |
|
| Submission date |
Jul 01, 2019 |
| Last update date |
Jul 27, 2020 |
| Contact name |
Jose Miguel Ramos Pittol |
| E-mail(s) |
Jose.Ramos-Pittol@uibk.ac.at
|
| Organization name |
University of Innsbruck
|
| Department |
Institute of Biochemistry
|
| Street address |
Innrain 80-82
|
| City |
Innsbruck |
| ZIP/Postal code |
6020 |
| Country |
Austria |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE133659 |
FXR isoform selective effects on hepatoma cell line HepG2 |
|
| Relations |
| BioSample |
SAMN12185835 |
| SRA |
SRX6387620 |
