Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Single flies frozen on dry ice were placed into each well of Axygen 96 Deep Well Plates (Corning, Corning, NY) pre-loaded with 200µL of 1 mm glass beads (Biospec Products, Bartlesville, OK). Fly placement in the wells of each plate was randomized within a replicated environment. We isolated total RNA using the RNeasy 96 Plate Kit according to the manufacturer using either vacuum or centrifugation technology (Qiagen, Valencia, CA), with the following modifications. We added 200µl RLT buffer to each well, sealed the plates with Axygen Sealing Mats (Corning, Corning, NY), and homogenized flies at room temperature for 30sec using a fixed-speed Mini Bead Beater (Biospec Products, Bartlesville, OK). We added 200µl of 70% ethanol to the crude homogenate, mixed by pipetting several times, and transferred the supernatant to RNeasy plates. We measured total RNA yield using the Quant-iTTM RiboGreen(R) RNA Assay Kit (Invitrogen, Carlsbad, CA) in black FLUORTRAC 600 384-well PS plates (Greiner Bio-One, Inc., Longwood, FL) using a Gemini EM Fluorescence Microplate Reader (Molecular Devices, Sunnyvale, CA). We used Dynabeads Oligo (dT) 25 (Life Technologies, Carlsbad, CA) to purify polyA RNA according to manufacturer instructions, except that we used 50-200ng of total RNA depending on yield, adjusted the volume to 50µl with distilled H2O, added 10µl of the washed Dynabeads in a 50µl binding buffer slurry, heated the samples at 65°C for 5min, and immediately chilled the samples on ice. To prepare RNA-Seq stranded libraries we modified an existing protocol (Wang et al., PLoS One, 2011, PMID 22039485). All steps were done in 96-well plate format. We incubated all reactions in a Tetrad PTC-225 Thermal Cycler (MJ Research, Waltham, MA). To clean up enzymatic reactions and size select 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 the protocol in (Rohland et al., Genome Res, 2012, PMID 22267522), except that we incubated samples with beads for 10 min and used 80% ethanol for washing. The volume of MagNA beads in XP buffer and corresponding concentration of PEG in the solution was 1.6× XP buffer for no size-selection (except where noted) and 1× XP buffer for selecting fragments > 200bp. Unless noted, we left the beads in the sample after each step. We used 96-well PCR plates (USA Scientific, Ocala, Fl) paired with Alpaqua 96R Ring Magnet Plates (Alpaqua, Beverly, MA) for magnetic bead separation. We measured RNA quantity with Quant-iTTM RiboGreen(R) and DNA with Quant-iTTM PicoGreen(R) (Invitrogen, Carlsbad, CA) in 384-well plates, PS, FLUOROTRAC 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 RNA degradation, library size (DNA), and adapter dimer contamination (DNA). We fragmented polyA RNA bound to Dynabeads at 94°C for 8min in 16µl of 1.25× 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., Genome Res, 2011, PMID 21816910) from pools 78A and 78B (Zook et al., PLoS ONE, 2012, PMID 22859977) obtained from Marc Salit (National Institute of Standards and Technology, Gaithersburg MD). We chilled the samples immediately on ice for 2min and eluted from the beads. We transferred 15µl of the eluate to a 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 distilled H2O. We then performed a second strand synthesis with dUTP by adding 5µl of 1× 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 instructions. We then rebound samples to MagNA beads, washed, and eluted as above. Next we performed dA-tailing by adding 4µl of 1× 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 distilled H2O, and transferred 10µl of cDNA to each of two fresh plates. One plate was used in the following steps and one plate was frozen as a back-up. We ligated RNA Adapter Indexes (Illumina, San Diego, CA) to dsDNA by adding 1µl of an adapter to each 10µl sample and 13µl of 1× 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 of MagNA beads in XP buffer (final PEG = 13.6%), bound cDNA to the beads, washed and eluted in 30µl of distilled H2O as above. We size-selected libraries by adding 1× XP buffer (30µl), washed, eluted samples with 24µl distilled H2O, and transferred 23µl of cDNA to a 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 2× 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 the product by adding 30µl MagNA beads in 1× XP buffer, eluted in 30µl distilled H2O and transferred 29µl of cDNA library 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 a strong signal in the 300-350 bp library target size and weak signal in the 100-150bp range (primer dimer products). Each 96-well plate was composed of 4 sets of samples with 24 unique indexes. We pooled equal amounts (5-15 ng) of each library into pools for multiplex sequencing. All multiplexed libraries were again quantified and checked for quality as above. Excess library pools were frozen for re-sequencing if required.