title: GSM9005955: FACS-sorted S phase Armc5 knock out mouse embryonic fibroblasts batch 1, P3; Mus musculus; RNA-Seq
library_construction_protocol: After an additional 1.5-hour culture, the cells were stained with 10 µM live dye Vybrant™ DyeCycle™ Violet (Invitrogen, V35003) in the culture medium for an additional 75 minutes at 37 °C. Approximately 1 x 10^6 G1-phase cells and 1 x 10^6 S-phase cells were sorted by fluorescence-activated cell sorting (FACS; BD FACSAria™ III). The total RNA of the sorted cells was extracted with Trizol in phase-lock tubes. The 4sU-labeled RNA was enriched according to a protocol by Biasini et al. with some modifications. Briefly, the total RNA was precipitated with isopropanol. The pellets were washed in 75% ethanol, resuspended in DEPC-treated H2O, and treated with DNase I (Invitrogen™ TURBO DNA-free™ Kit) to eliminate genomic DNA. 4-sU-labeled RNA was biotinylated with EZ-link HPDP-Biotin (Thermo Fisher, 21341) and subsequently purified using phenol: chloroform: isoamyl alcohol (25:24:1, v/v; Invitrogen, 15593031). RNA was precipitated with isopropanol and NaCl, and the pellets were washed twice with 75% ethanol. Biotinylated RNA was captured on streptavidin magnetic beads (New England Biolabs, S1420S), which were washed sequentially with binding (0.5 M NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA) and ice-cold low-salt washing buffer (0.15 M NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA). Beads were resuspended in 100 µl water containing freshly prepared 100 mM DTT and incubated at room temperature for 1 minute to elute the biotinylated RNA. The eluted RNA was then purified with Qiagen RNeasy Mini Kit according to the manufacturer's instructions and finally eluted in nuclease-free water. The KAPA RNA HyperPrep Kit with RiboErase (HMR) module (Roche Diagnostics) was used for ribodepletion and library preparation for 123 ng 4-sU-labeled RNA. Library distribution was assessed using a 2100 Bioanalyzer (Agilent Technologies), and the libraries were quantified by quantitative PCR (qPCR). Libraries were sequenced as paired-end readcounts (PE100) on a Novaseq 6000 system (Illumina) using an S4 flow cell with 50 million readcounts per library.