Doxo_1d_2
Homo sapiens
Protocols: Once the full treatment (if applicable) was performed, cells were collected in 350 μl of RNA Lysis buffer RLY (Bioline, BIO-52079) and 3.5 μl of β-mercaptoethanol using a cell scraper. Samples were stored at -80C until processed. Human foreskin fibroblasts (BJ) were purchased from ATCC (Cat: CRL-2522). The cells were cultured in DMEM (Thermo Fisher Scientific) enriched with 10% fetal bovine serum (FBS, GE Healthcare Life Sciences) and 1% penicillin/streptomycin (Lonza) in 5% oxygen incubator. Cells were tested regularly for mycoplasma infection. Cells were treated either once for 24 hours with 250 nM doxorubicin (Tebu-bio, BIA-D1202-1), or 8 times for 24 hours in consecutive days with 1 μM abemaciclib (MedChem Express, HY-16297). In all drug-treated cases, RNA was collected at both 1 day and 8 days post drug removal. Cells treated 8 times for 24 hours with the vehicle alone (water in volumes equal to the ones used for palbociclib or abemaciclib) were used as control and RNA was collected at the end of the treatment. Raw sequencing data were demultiplexed according to library-specific barcodes and converted to fastq format using standard Illumina software (bcl2fastq version 1.8.4). Total RNA was extracted from cells via a ISOLATE II RNA mini kit (Bioline, Cat: BIO-52073) following manufacturer's instructions. The extracted RNA was quantitated using a Nanodrop and RNA quality was measured via BioAnalyzer RNA chip (Agilent). Poly-A tail selection was used to enrich for messenger RNA (mRNA) using the NEXTflex Poly(A) Beads kit (Bioo Scientific Corp, cat #512980). RNA-Seq library preparation was carried out using NEXTflex Rapid Directional qRNA-Seq kit (Bioo Scientific, cat# 5130-05D). In brief, mRNA was fragmented using a cationic buffer and then submitted to first and second strand synthesis, followed by adenylation, ligation and 15 cycles of PCR. Library quality and size distribution were validated on a Qubit (Thermo Fisher Scientific) and an Agilent High Sensitivity DNA chip.
ENA-FIRST-PUBLIC
2020-01-31T04:02:40Z
ENA-LAST-UPDATE
2019-01-31T16:01:37Z
External Id
SAMEA5313056
INSDC center name
European Research Institute for the Biology of Ageing (ERIBA) , University of Groningen, University Medical Center Groningen
INSDC first public
2020-01-31T04:02:40Z
INSDC last update
2019-01-31T16:01:37Z
INSDC status
public
Submitter Id
E-MTAB-7642:Doxo_1d_2
broker name
ArrayExpress
cell line
BJ
cell type
fibroblast
common name
human
developmental stage
neonate
disease
normal
organism part
foreskin
sample name
E-MTAB-7642:Doxo_1d_2
scientific_name
Homo sapiens
sex
male