GSM6248559: AL1 cells, DMSO, L78078; Ambystoma mexicanum; RNA-Seq
For RNA-seq analysis of AL1 cells, senescent or proliferating control cells in 10 cm dishes were harvested at 14 days post induction. Briefly, cells were rinsed twice with 0.8X PBS, trypsinised, and pelleted. Subsequently, pellets were lysed in 350 ml b-mercaptoethanol-supplemented RLT+ buffer. Total RNA was then extracted using RNeasy micro plus RNA extraction kit (Qiagen) according to manufacturer's instructions. 1000 ng of total RNA was used as input for library preparation. Processing of the samples for RNA-sequencing was done following  a modified Smartseq-2 protocol (https://star-protocols.cell.com/protocols/1595). In brief, samples were thawed on ice and we added 0.5ul of dT-buffer to each sample, containing poly-dT oligos (final concentration of 0.5uM) for capturing mRNAs. Subsequently, we performed reverse transcription and cDNA amplification. Libraries were prepared with the Illumina DNA Prep Tagmentation kit and sequenced on an Illumina NovaSeq 6000 to an average of ~40mio PE100bp fragments/sample.