GSM1648178: S3 inactive Chemotype 2 with DMSO and oleic acid 4hrs rep 2; Drosophila melanogaster; RNA-Seq
The plates were removed from the -80°C freezer, and RNA was extracted with the RNeasy Mini Kit (Qiagen, West Sussex, UK) according to the manufacturer's instructions. In brief, the cells were resuspended in 600µL of the RLT buffer provided with the kit. The RLT buffer was supplemented with 40µL of a 1M DTT solution per mL of RLT buffer. Otherwise, the standard protocol was followed and RNA was eluted by 2 times 30µL. Purified total RNA was stored at -80°C until further use. Dynabeads Oligo (dT)25 (Life Technologies, Carlsbad, CA) were used to purify mRNA according to manufacturer instructions, except that we used 200ng of total RNA adjusted the volume to 50µl with dH2O, added 10µl of the washed Dynabeads in a 50µl binding buffer slurry, heated the samples at 65°C for 5min and immediately chilled on ice. To prepare RNA-Seq stranded libraries we modified an existing protocol (Wang et al., 2011). All steps were done using 96-well PCR plates (USA Scientific, Ocala, Fl). We incubated all reactions in a Tetrad PTC-225 Termal Cycler (MJ Research, Waltham, MA). To clean up enzymatic reactions and size select library fragments at any of several steps below, we used 0.1% w/v carboxyl-modified Sera-Mag Magnetic Speed-beads (MagNA beads, Thermo Fisher Scientific, Waltham, MA) in XP buffer [20% PEG 8000, 2.5 M NaCl, (Sigma Aldrich, St. Louis, MO)] following protocol (Rohland and Reich, 2012), except that we incubated samples with beads for 10 min and used 80% ethanol for washing. For cleaning without size-selection we used 1.6X MagNA beads in XP buffer (except where noted) and for selecting fragments > 200bp - 1X MagNA beads in XP buffer. Unless noted, we left the beads in the sample after each step. We used Alpaqua 96R Ring Magnet Plates (Alpaqua, Beverly, MA) for magnetic bead separation. We measured RNA quantity with Quant-iTª RiboGreen and DNA with Quant-iTª PicoGreen (Invitrogen, Carlsbad, CA) in 384-well plates, PS, Fluotrac 600, black (Greiner Bio-One Inc, Longwood, Fl) using a Gemini EM Fluorescence Microplate Reader (Molecular Devices, Sunnyvale, CA) according to manufacturer instructions. We used Agilent Bioanalyzer RNA chips and High Sensitivity DNA chips on the 2100 Bioanalyzer system (Agilent, Santa Clara, CA) according to manufacturer instructions and visually inspected electrophoregrams for sharp peaks for simple pass/fail calls based on signs of obvious degradation and to check for library size and adapter dimer contamination. We fragmented mRNA bound to Dynabeads at 94°C for 8min in 16µl of 1.25X first strand MMuLV RT buffer (New England Biolabs, Beverly, MA), with 100ng random primers (Invitrogen, Carlsbad, CA), and 10pg ERCC spike-in controls (Jiang et al., 2011) from pools 78A and 78B (Zook et al., 2012) obtained from Marc Salit (NIST, National Institute of Standards and Technology, Gaithersberg MD). We chilled the samples immediately on ice for 2min and eluted from the beads. We transferred 15µl of the eluate to fresh 96-well PCR plate. We added 5µl of the first strand synthesis mixture [0.3mM dNTPs, 5mM DTT, and 10U M-MuLV RT (New England Biolabs, Beverly, MA), and 0.5U SuperRase-In (Life Technologies, Carlsbad, CA)] to the fragmented RNA in the fragmentation buffer and performed a reverse transcription reaction. We bound RNA/cDNA hybrid with 32µl MagNA bead XP buffer slurry, washed, and eluted samples in 16µl of dH2O. We then performed a second strand synthesis with dUTP by adding 5µl of 1X NEB buffer2 (New England Biolabs, Beverly, MA), with 1mM each of dATP, dCTP, dGTP and 2mM dUTP (Thermo Fisher Scientific, Waltham, MA), 10U DNA PolI, 2.5U RNAseH, and 2.5mM DTT (New England Biolabs, Beverly , MA) and incubated at 16°C for 5 hours. We rebound, washed, and eluted as above. We repaired ends by adding 4µl of NEBNext End Repair Module (New England Biolabs, Beverly, MA) to the eluate following manufacturer instruction. We then rebound samples to MagNA beads, washed, and eluted as above. Next we performed dA-tailing by adding 4µl of 1X Blue Buffer, with 1U Klenow 3’-5’ exo- (Enzymatics, Beverly, MA) and 1mM dATP, to the eluate and incubated at 37°C for 30min. We then rebound samples to MagNA beads and washed as above, eluted with 21µl dH2O, and transferred 10µl of cDNA without beads to each of two fresh plates. One plate was used in the following and one plate was frozen as a back-up. We ligated RNA Adapter Indexes AR001-AR016, AR018-AR023, AR025, and AR027 (Illumina, San Diego, CA) to dsDNA by adding 1µl of an adapter to each 10µl sample and 13µl of 1X Rapid Ligation Buffer with 30U T4 DNA Ligase (Enzymatics, Beverly, MA), and incubated at 25°C for 10min. We stopped reactions with a final concentration of 0.01M EDTA. We added 25µl MagNA beads XP buffer slurry (final PEG = 13.6%), bound cDNA to the beads, washed and eluted in 30µl of dH2O as above. We size-selected libraries by adding 1X XP buffer (30µl), washed, eluted samples with 24µl dH2O, and transferred 23µl of cDNA without beads to fresh plate. 11.5µl of the eluate was transferred to another plate and used in the next step; the remaining samples were frozen as a back-up. We mixed dsDNA product with 2.5U (0.5µl) of Uracil DNA Glycosylase (New England Biolabs, Beverly, MA) and 3µl of PCR Primer Cocktail (Illumina, San Diego, CA) and incubated at 37°C for 30min to digest the second strand DNA. We added 15µl 2X KAPA HiFi HotStart ReadyMix (Kapa Biosystems, Woburn, MA) directly to the UDG digested DNA mixture and performed a PCR amplification with the following programmed cycle: 95°C for 2min, followed by 12 cycles of 98°C for 20sec, 65°C for 30sec, and 72°C for 30sec; then 72°C for 5min. We purified product by adding 30µl MagNa beads XP buffer slurry, eluted in 30µl dH2O and transferred 29µl of cDNA library without beads to a fresh plate as described above. To assay plate-level failure, we took 11 samples from each 96-well plate and examined electropherograms for the strong signal in the 300-350 bp library target size and weak signal in the 100-150bp range (primer dimer products).