Brachypodium distachyon transcriptome SMART
Total RNA was isolated using modified protocol previously described (Filichkin et al., 2007). First, RNA was extracted using “The Plant RNA Reagent” (Invitrogen). RNA was treated for 10 min at 65°C with RNAsecure reagent (Ambion). To eliminate genomic DNA amplification, all RNA preparations were treated for 15 min at 37°C with RNase-free “Turbo DNase” (Ambion). Next, total RNA was further purified using “RNAeasy Mini RNA kit” (Qiagen) according to the manufacturer’s RNA clean up protocol. For the RP libraries the poly(A) mRNA essentially free of ribosomal and other non-polyadenylated RNAs was isolated by two consequent cycles of purification on oligo d(T) cellulose using “Micro-PolyA-Purist” kit (Ambion). Concentration, integrity and extent of contamination by ribosomal RNA were monitored using ND-1000 spectrophotometer (Thermo Fisher Scientific) and Bioanalyzer 2100 (Agilent Technologies). To increase transcript representation, RNA from different tissues/samples was combined in equal proportion prior to generation of cDNA libraries. FL libraries were prepared essentially as described (Fox et al., 2009). Libraries were generated using the SMART method (Zhu et al., 2001). cDNA (5-7 µg in a 50 µL volume) was mixed with 750 µL of Illumina nebulization buffer and fragmented for 7 min in a nebulizer (Invitrogen) using compressed nitrogen at 35 psi. The sheared cDNA was purified using “QIAquick PCR Purification Kit” (Qiagen) and eluted into 32 µL of water. For the RP libraries, the first cDNA strand was synthesized using 1 µg of poly(A) mRNA essentially free of rRNA, random hexamer primers (300 ng per each µg of RNA), and Superscript III reverse transcriptase (RT) (Invitrogen). The second strand of cDNA was synthesized using DNA polymerase I (Klenow fragment) by combining 20 ?L of the 1st strand reaction, 8 ?L of 10x Klenow Buffer (NEB), 1 unit of RNAse H (Invitrogen), 68.8 ?L of water and 30 units of DNA Polymerase I (NEB). The reaction was incubated for 90 min at 15oC and cDNA was purified using “Qiaquick PCR clean up” kit. FL or RP cDNA (30 µL) was combined with 10 µL of 10 mM ATP in 5X T4 DNA ligase buffer (Invitrogen), 4 µL of 10 mM dNTPs mix, 2.5 µL of T4 DNA polymerase (3 U/µL), 1 µL of Klenow DNA Pol (5 U/µL) and 2.5 µL of T4 polynucleotide kinase (10 U/µL, NEB). After incubation for 30 min at 20ºC the DNA was purified using “QIAquick PCR Purification kit”. To add dA to the termini, 32 µL DNA from the prior step was mixed with 5 µL of 10X Klenow buffer, 10 µL of 1 mM dATP, and 3 µl of Klenow exo-polymerase (3’ to 5’ exo minus, 5 U/µL, NEB) and incubated for 30 min at 37oC. DNA was purified using a “QIAquick MinElute Reaction Clean-up” (Qiagen) kit and eluted into 12 µL of water. To ligate Illumina adapters, 10 µL of cDNA from the prior step was mixed with 5 µL of 5X T4 DNA ligase buffer, 6 µL of adapter oligo mix, 4 µL of T4 DNA ligase (NEB) and incubated for 15 min at 25oC. DNA was purified using a “QIAquick MinElute PCR Purification Kit”. cDNA was size-fractionated (typically with average fragment length of 170-250 base pairs) on 3.5% (w/v) “NuSieve GTG” agarose (Lonza). The fractionated libraries were PCR amplified using “Phusion Hot Start High-Fidelity” DNA polymerase (NEB) and the following PCR protocol: 30 sec at 98oC, then 10 sec at 98oC, 30 sec. at 65oC, and 30 sec. at 72oC for 18 cycles, followed by a 10-min extension step at 72oC. Prior to cluster generation, DNA was diluted to a final concentration of 5 to 10 pM to generate 25,000 to 40,000 clusters per tile of the flow cell.