description: Protocols: Cells were seeded in 150-mm cell culture dishes and were treated with alisertib or etoposide for 7 days. Senescent cells were subsequently cultured for 24 hours without drug. Control cells were grown in 150-mm cell culture dishes and were harvested at 80% confluency. Total RNA was isolated using the RNeasy Mini Kit (74106, Qiagen), including an on-column DNase digestion (79254, Qiagen), according to the manufactures instructions. Quality and quantity of the total RNA was assessed by the 2100 Bioanalyzer using a Nano chip (Agilent, Santa Clara, CA). Total RNA samples having RIN>8 were subjected to library generation. Strand-specific libraries were generated using the TruSeq Stranded mRNA sample preparation kit (Illumina Inc., San Diego, RS-122-2101/2) according to the manufacturer's instructions (Illumina, Part # 15031047 Rev. E). Briefly, polyadenylated RNA from intact total RNA was purified using oligo-dT beads. Following purification the RNA was fragmented, random primed and reverse transcribed using SuperScript II Reverse Transcriptase (Invitrogen, part # 18064-014) with the addition of Actinomycin D. Second strand synthesis was performed using Polymerase I and RNaseH with replacement of dTTP for dUTP. The generated cDNA fragments were 3' end adenylated and ligated to Illumina Paired-end sequencing adapters and subsequently amplified by 12 cycles of PCR. The libraries were analyzed on a 2100 Bioanalyzer using a 7500 chip (Agilent, Santa Clara, CA), diluted and pooled equimolar into a multiplex sequencing pool.
attributes.tag: ENA first public
attributes.value: 2021-07-18
attributes.tag: ENA last update
attributes.value: 2021-07-18
attributes.tag: External Id
attributes.value: SAMEA7746884
attributes.tag: INSDC center alias
attributes.value: NETHERLANDS CANCER INSTITUTE
attributes.tag: INSDC center name
attributes.value: NETHERLANDS CANCER INSTITUTE
attributes.tag: INSDC first public
attributes.value: 2021-07-18T20:39:18Z
attributes.tag: INSDC last update
attributes.value: 2021-07-18T20:39:18Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9970:A549_Ali7_batch3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: A549
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: non-small cell lung carcinoma
attributes.tag: sample name
attributes.value: E-MTAB-9970:A549_Ali7_batch3
attributes.tag: sequencing batch
attributes.value: 3