title: GSM5740342: RATE-seq Neu RTT 4h rep1; Homo sapiens; RNA-Seq
library_construction_protocol: After 4sU incorporation, cells were quickly washed twice with ice-cold 1× PBS Total RNA and scraped into ice-cold 1.5 ml Eppendorf tubes.  Cells were collected by spinning at 1000g for 5 min at 4°C and cell pellets were resuspended in 1 mL of Trizol (Thermo Fisher Scientific). Total RNA was extracted according to manufacturer instructions. 50 µg of total neuronal RNA was mixed with 5 µg unlabeled yeast RNA and 5 µg 4sU-labeled S2 fly RNA in a total volume of 120 µL. 1 mg/mL HPDP-biotin (ThermoFisher Scientific) was reconstituted in dimethylformamide by shaking at 37°C for 30 min at 300 RPM. 120 µL of 2.5× citrate buffer (25 mM citrate, pH 4.5, 2.5 mL EDTA) and 60 µL of 1 mg/mL HPDP-biotin were added to the premixed RNA sample for each time point. The solution was incubated at 37°C for 2 h at 300 RPM on an Eppendorf ThermoMixer F1.5 in the dark. Samples were extracted twice with acid phenol, pH 4.5, and once with chloroform. RNA was precipitated with 18 µL 5M NaCl, 750 µL 100% ethanol, and 2 µL GlycoBlue (Invitrogen) overnight at −20°C. Precipitated RNA was pelleted for 30 min at 21,000g at 4°C. The RNA pellet was resuspended in 200 µL of 1× wash buffer (10 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM EDTA). Biotinylated RNA was purified using the µMACS Streptavidin microbeads system (Miltenyi Biotec). 50 µL Miltenyi beads per sample were pre-blocked with 48 µL 1× wash buffer and 2 µL yeast tRNA (Invitrogen), rotating for 20 min at room temperature. µMACS microcolumns were washed 1× with 100 µL nucleic acid equilibration buffer (Miltenyi Biotec), followed by 5× washes with 100 µL 1× wash buffer. Beads were applied to microcolumns in 100 µL aliquots and again washed 5× with 100 µL 1× wash buffer. Beads were demagnetized and eluted off the column with 2× 100 µL 1× wash buffer, and columns were placed back on the magnetic stand. A total of 200 µL beads was mixed with each sample of biotinylated RNA and rotated at room temperature for 20 min. Samples were applied to the microcolumns in 100 µL aliquots, washed 3× with 400 µL wash A buffer (10 mM Tris-HCl, pH 7.4, 6 M urea, 10 mM EDTA) prewarmed to 65°C, and washed 3× with 400 µL wash B buffer (10 mM Tris-HCl, pH 7.4, 1 M NaCl, 10 mM EDTA). RNA was eluted with 5× 100 µL of 1× wash buffer supplemented with 0.1 M DTT, and flow-through was collected in a tube. Purified RNA was precipitated with 30 µL 5 M NaCl, 2 µL GlycoBlue, and 1 mL 100% ethanol, incubated at −20°C overnight. Samples were spun at 21,000g at 4°C for 30 min and resuspended in 20 µL water. RNA quality was assessed by running 3 µL of samples on a 1.5% agarose gel. RNA-seq libraries were prepared for each time-points and seady-state sample using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen) automated on the NGS WorkStation (Agilent) at The Centre for Applied Genomics (TCAG) according to the manufacturer's instructions. PCR cycle numbers were determined using the PCR Add-on Kit for Illumina (Lexogen). All steady-state samples were processed with 250 ng of total RNA input. To minimize variability between time-points within a batch, RNA samples were processed with the same total RNA input with a minimum of 100 ng of total RNA used. Each sample was spiked-in with ERCC RNA Spike-In Control Mix 1 (Ambion) according to the manufacturer's instructions prior to the start of library preparation. Library quality and quantity were measured at TCAG prior to sequencing with Bioanalyzer (Agilent) and KAPA qPCR (Roche). Sequencing was also performed at TCAG on the Illumina HiSeq 2500 with single-end 100bp read length yielding 40 to 50 million reads.