title: NextSeq 500 paired end sequencing; RNA-seq of rRNA-depleted total RNAs in WI-38hTERT/sh3-H2AFJ and WI-38hTERT/sh-NT fibroblasts in proliferation and etoposide-induced senescence.
design: RNA-seq of rRNA-depleted total RNAs in WI-38hTERT/sh3-H2AFJ and WI-38hTERT/sh-NT fibroblasts in proliferation and etoposide-induced senescence.
library_construction_protocol: Cells were trypsinized, washed once with PBS, and lysed in mirVana lysis buffer.  WI38 human embryonic fibroblasts from the ATCC were immortalized with hTERT and grown in MEM (Invitrogen #21090) + 10% Fetal Bovine Serum + 1 mM Sodium Pyruvate + 2 mM L-glutamine + 0.1 mM non-essential amino acids + pen/strep in an incubator with 5% C02 and 5%02. WI38hTERT fibroblasts cells were grown in the presence of 1 µg/ml doxycycline for 3 days to induce the expression of sh-RNAs before senescence induction by incubation with 100 µM etoposide for 48 hours. Cells were then washed and incubated in fresh medium without etoposide for 7 days before harvesting cells for analysis. Doxycycline was maintained throughout the experiment to ensure continual expression of the sh-RNAs. RNA was purified with the mirVana miRNA isolation kit (Thermo AM1560). The total RNA fraction was used for library construction. RNA was purified using the mirVana miRNA Isolation Kit (ThermoFisher Scientific, #AM1560) according to the manufacturer's protocols to yield a fraction containing total RNAs and a second fraction enriched in small RNAs. The total RNA fraction was used with the Illumina Ribo-Zero Scriptseq library kit to deplete cytosolic rRNA and prepare DNA librairies.