title: NextSeq 500 paired end sequencing; RNA-seq of small RNAs in WI-38hTERT/sh3-H2AFJ and WI-38hTERT/sh-NT fibroblasts in proliferation and etoposide-induced senescence
design: RNA-seq of small 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 small RNA fraction was used for library construction. The small RNA fraction was used with the NEBNext small RNA library prep kit (New England Biolabs, #E7300) to produce a small RNA library. Libraries were subjected to size selection on a 6% native polyacrylamide gel to enrich for molecules with inserts within the miRNA size range (18-25 nt).