Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Following PBS washes, cell pellets were lysed in either TRI Reagent (Sigma-Aldrich) or Isol-RNA Lysis Reagent Manual (5Prime), according to manufacturer's instructions. Briefly, 100 µL of 1-bromo-3-chloropropane was added to each sample, and samples were vortexed for 15 seconds and incubated at room temperature for 2 minutes. Sample were centrifuged in a prechilled centrifuge at 4°C at 15500 x g for 15 min. Aqueous phase was transferred to a new tube and 500µL isopropanol and 2µL glycoblue were added. Samples were vortexed for 10 s and then incubated for 10 min at room temperature. Samples were then centrifuged at 4°C at 15500 x g for 10 min. Supernatant was discarded and pellet was washed with 1 mL of 75% ethanol and centrifuged at 4°C, 9000 x g for 5 min. Ethanol was removed and pellet was resuspended in 20 µL Milli-Q water. For Saccharomyces cerevisiae 10 ml of the liquid culture was pelleted for 3 min at 1500 x g at 4°C. Supernatant was discarded and pellet was resuspended in 1 ml of ice-cold milli-Q water and pelleted again. The cell pellet was resuspended in 400 µL TES solution (10 mM Tris-Cl pH 7.5, 10mM EDTA, 0.5% SDS). 400 µL Phenol:Chloroform 5:1 was added, and the sample was vortexed vigorously for 10 s. After incubating at 65°C for 60 min with periodic vortexing, the sample was then centrifuged for 5 min at 21000 x g at 4°C. The aqueous phase was transferred to a new tube and reextracted with acid phenol and then again with chloroform. The aqueous phase was transferred, and RNA was precipitated with 40 µL of 3M sodium acetate, pH5.3 and 1 mL of ice-cold 100% ethanol. RNA was pelleted at 21000 x g for 5 min at 4°C. The RNA pellet was washed with ice-cold 70% ethanol and resuspended in 50 µL of milli-Q water. For Caenorhabditis elegans, sample volumes were brought up to 100 µL with UltraPure water and resuspended in 4X volume of TRI-Reagent. Samples were vortexed for 10 minutes at room temperature. 10% volume of 1-bromo-3-chloropropane was added and samples vortexed for an additional 10 minutes at room temperature. Samples were then centrifuge for 8 minutes at 10,000 x g at 4 degrees and top aqueous layer was transferred to a new tube. RNA was precipitated with 20 µg glycogen and 4X volume of 100% ethanol for at least 1 hour at -20°C. For transcription inhibition samples, 4 µg sample RNA was combined with 10 % Saccharomyces cerevisiae RNA. For metabolically labeled samples a 1 mg/mL solution of HPDP-biotin (Thermo Fisher Scientific) in dimethylformamide was incubated at 37°C for 30 minutes. 40-100 µg of RNA was combined with 20% w/w unlabeled yeast RNA (C. elegans N2 RNA for 293_not_enough samples gifted by the Claycomb lab), 20% w/w 4SU-labeled fly RNA and 120 µL of 2.5x citrate buffer (25 mM citrate pH 4.5, 2.5 mM EDTA) in a total volume of 240 µL. 60 µL of HPDP-biotin solution was added, and the solution was incubated at 37°C for 2 hr, covered and shaking at 200 rpm. Equal volume acid phenol was added, and samples were vortexed and separated at 21000 x g for 5 min at 4°C. Aqueous layer was re-extracted with acid phenol and again with chloroform. RNA was precipitated with 18 µL of 5M NaCl, 750 µL of 100% ethanol, and 2 µL glycoblue for a minimum of 60 min at –20°C. RNA was pelleted for 30 min at 21000 x g at 4°C and supernatural discarded. RNA pellet was resuspended in 200 µL 1x wash buffer (10 mM Tris-Cl pH 7.4, 50 mM NaCl, 1 mM EDTA). 50 µL MACS microbeads (Miltenyi Biotec) were incubated with 48 µL of 1x wash buffer and 2 µL yeast tRNA for 20 min at room temperature with rotation. The microcolumns (Miltenyi Biotec) were washed with 100 µL nucleic acid equilibration buffer (Miltenyi Biotec) and then five times with 100 µL 1x wash buffer. Beads were applied to the column in 100 µL aliquots and washed five times with 100 µL 1x wash buffer. Columns were demagnetized, and beads eluted with two 100 µL washes with 1x wash buffer. 200 µL of bead solution was combined with RNA sample and rotated at room temperature for 20 min. Sample was then applied to the remagnetized column in 100 µL aliquots. Columns were washed three times with 400 µL of wash 1 buffer (10mM Tris-Cl pH 7.4, 6M urea, 10mM EDTA) prewarmed to 65°C and then three times with 400 µL wash 2 buffer (10 mM Tris-Cl pH 7.4, 1M NaCl, 10mM EDTA). Selected RNA was eluted with five washes of 1x wash buffer with 0.1M dithiothreitol. RNA was precipitated for at least 1 hr at –20°C with 30 µL 5M NaCl, 1 mL ethanol, and 2 µL glycoblue and then pelleted for 30 min at 21000 x g and 4°C. Sequencing libraries were prepared using the TruSeq Stranded mRNA Sample preparation kit (Illumina) per the manual (Rev. E).