MaleBrainIllumina2.5
RNA extraction:   Half of a vole male brain was divided into 3 approximately 50-100mg sections and stored in RNAlater (Ambion AM7020). RNA was extracted using TRIzol Plus RNA Purification Kit (Invitrogen 12183-555) including an on column DNase digestion using PureLink DNase (Invitrogen 12185-010).  The RNA from the three brain sections was pooled and quantitated using nanodrop then diluted 1:250 in TE and quality checked via use of an Agilent Pico Chip (Agilent 5067-1513).    mRNA purification:  The mRNA was purified using Illumina’s mRNA Sequencing Protocol Rev. D with Illumina’s Kit Contents Bag 1 (part 1004815) and Kit Contents Box 1 (part 1004814).  To purify mRNA, 2.5ug of starting total RNA were diluted to 50uL with water and incubated at 65 degrees Celsius for 5 minutes then placed on ice.  Then 15uL of Sera-Mag oligo(dT) were prepared in 1.5mL tubes by washing twice with 100uL Bead Binding Buffer and resuspending in 50uL Bead Binding Buffer.  To the RNA 50uL beads in buffer were added and rotated at room temperature for 5 minutes and the supernatant was discarded.  The beads were washed twice with 200uL Wash Buffer.  For the first elution, 50uL of 10mM Tris-HCl were added to the beads and incubated at 80 degrees Celsius for 2 minutes then immediately placed on magnetic stand.  The supernatant containing RNA was transferred to a new tube and placed at 65 degrees Celsius for 5 minutes.  The beads were then washed twice with 200uL wash buffer and placed on ice.  To bind the mRNA, the RNA was then added to the beads and rotated at room temperature for 5 minutes and the supernatant was discarded. Then the beads were washed twice with 200uL Wash Buffer.  For the second elution, 17uL of 10mM Tris-HCl was added to the beads and incubated at 80 degrees Celsius for 2 minutes then immediately placed on magnetic stand.  The supernatant for was then transferred to a 200uL PCR tube.  mRNA fragmentation:  To the mRNA, 4uL 5Xfragmentation Buffer was added and incubated at 94 degrees Celsius for 5 minutes.  Then 2uL Fragmentation Stop Solution was added and placed on ice.  The sample was transferred to a 1.5mL tube and precipitated with 2uL 3M NaOAC pH 5.2, 2uL glycogen, and 60uL 100% ethanol and incubated at -80 degrees Celsius for 30 min then spun at 4 degrees Celsius for 25 minutes at 14K rpm.  The supernatant was removed carefully with a pipette so as not to disturb the pellet. The sample was then washed with 300uL 70% ethanol and resuspended in 11.1uL RNase free water.  First Strand Synthesis:  For the First strand synthesis, 1uL Random Primers and 11.1uL mRNA template were combined and incubated at 65 degrees Celsius for 5 minutes then placed on ice.  In a separate tube 4uL 5X First Strand Buffer, 2uL 100mM DTT, 0.4uL 25mM dNTP mix and 0.5uL RNase Inhibitor were combined, then added and incubated for 2 minutes at 25 degrees Celsius.  Then 1uL of SuperScript II (Invitrogen 18064-014) was added and incubated at 25 degrees Celsius for 10 minutes, 42 degrees Celsius for 50 min, 70 degrees Celsius for 15 min, held at 4 degrees Celsius, and finally placed on ice.  Second Strand Synthesis:  For the second strand synthesis, 62.8uL of water was added to the first strand synthesis cDNA template.  Then 10uL GEX Second Strand Buffer and 1.2uL 25mM dNTP mix were added to the template and incubated on ice for 5 minutes after which 1uL RNase H and 5uL DNA Pol were added and incubated at 16 degrees Celsius in for 2.5 hours. The sample was purified using the QIAquick PCR Purification Kit (Qiagen 28104) and eluted in 50uL QIAGEN EB buffer each.  The sample was stored at -20 degrees Celsius then handed to Emory’s Genomics Core Facility.  To repair the ends of product, 50uL of the eluted DNA, 27.4uL water, 10uL 10X End Repair Buffer, 1.6uL 25mM dNTP Mix, 1uL Klenow DNA Polymerase, 5uL T4 PNK were combined, and incubated at 20 degrees Celsius for 30 minutes. The sample was then eluted in 32uL Qiagen EB buffer using the Qiagen  QIAquick PCR Purification Kit. The eluted DNA is then combined with 5uL A-Tailing Buffer, 10uL 1mM dATP, and 3uL Klenow exo to add an ‘A’ base to the blunt DNA product.  The sample is then incubated at 37 degrees Celsius for 30 minutes and purified using the QIAquick PCR Purification Kit and eluted in 23uL buffer EB.   In order to ligate the adapters to the DNA fragments, 25uL 2X Rapid T4 DNA Ligase Buffer, 1uL PE Adapter Oligo Mix, and 1ul T4 DNA Ligase per reaction are added to the 23uL DNA and incubated at room temperature for 15 minutes.  The sample was purified using the MinElute PCR Purification Kit (QIAGEN 28004) and eluted in 10uL of the EB buffer.   The sample was run on the Invitrogen E gel (G7208-02) 2% agarose Size Select system, eluted in 20 ul EB buffer.  To enrich the template 10uL of 5X Phusion Buffer, 1uL of PCR Primer PE 1.0 (5' AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T), 1uL of PCR Primer PE 2.0 (5' CAA GCA GAA GAC GGC ATA CGA GAT CGG TCT CGG CAT TCC TGC TGA ACC GCT CTT CCG ATC T), 0.5uL of 25mM dNTP, 0.5uL of Phusion DNA Polymerase, and 7uL of water were added to a 200uL PCR tube.  Then 1uL of the purified sample plus 19uL water were added to the mixture and cycled using an initial incubation of 98 degrees Celsius for 30 seconds, followed by 15 cycles of 98 degrees Celsius for 10 seconds, 65 degrees Celsius for 30 seconds, and 72 degrees Celsius for 30 seconds, and  finishing with a final extension at 72 degrees Celsius for 5 minutes holding at 4 degrees Celsius at the end of the program. The PCR product was purified using Ampure XP beads (Beckman Coulter Genomics, A63880).  1.8X volumes of AMPure XP were added  to the PCR reaction, mixed thoroughly, and incubated for 5 minutes at room temperature (8 to 30 degrees Celsius).  The tube was placed onto a a  low - or medium - throughput magnet stand for 2 minutes to separate the paramagnetic particles from the solution and the supernatant was removed and discarded. To wash the sample, 200uL of 70% ethanol was added to the tube and incubated at room temperature (8 to 30 degrees Celsius) for a minimum of 30 seconds then removed. This step was repeated for a total of 2 ethanol washes.  To elute the sample, the tube was removed from the magnetic stand and 40uL of elution buffer (reagent - grade water, TRIS acetate with a pH of 8.0, or TE) was added and mixed.  The sample was incubated at room temperature (8 to 30 degrees Celsius) for 2 minutes.  To separate the paramagnetic particles from the solution the tubes were placed onto the magnetic stand for 1 minute.  The purified product was then transferred to a new tube and validated using the Agilent Hight Sensitivity Chip (G2938-90320). Note, Illumina adapter-primer sequence, AGATCGGAAGAGCGGTTCAGCAGGAA,  is present at a the 3-prime end of some fraction of the sequenced reads presumably due to short insert sizes.