Heat_Stress_38
Total RNA was extracted using “The Plant RNA Reagent” (Invitrogen). RNA was treated for 10 min at 65C with RNAsecure reagent (Ambion). To eliminate genomic DNA amplification, all RNA preparations were treated for 15 min at 37C with RNase-free “Turbo DNase” (Ambion). Next, total RNA was further purified using the “RNAeasy Mini RNA kit” (Qiagen) according to the manufacturer’s RNA clean up protocol. Isolation of mRNA essentially free of ribosomal and other non-polyadenylated RNAs was critical for generation of non-biased randomly primed (RP) libraries. For the RP libraries the poly(A) mRNA was isolated by two consecutive cycles of purification on oligo d(T) cellulose using the “Micro-PolyA-Purist” kit (Ambion). Concentration, integrity and extent of contamination by ribosomal RNA were monitored using a ND-1000 spectrophotometer (Thermo Fisher Scientific) and Bioanalyzer 2100 (Agilent Technologies). Full-length enriched (FL) cDNA libraries were generated using the SMART method (Zhu et al., 2001). cDNA (5-7 ug in a 50 µL volume) was mixed with 750 uL 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 uL of water. For the randomly primed (RP) cDNA libraries the first cDNA strand was synthesized using 1 ug of poly(A) mRNA essentially free of rRNA, random hexamer primers (300 ng per each ug 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 uL of the 1st strand reaction, 8 uL of 10x Klenow Buffer (NEB), 1 unit of RNAse H (Invitrogen), 68.8 uL of water and 30 units of DNA Polymerase I (NEB). The reaction was incubated for 90 min at 15C and cDNA was purified using “Qiaquick PCR clean up” kit. To prepare cDNAs for sequencing on the Illumina Genome Analyzer, FL or RP cDNA (30 uL) was combined with 10 uL of 10 mM ATP in 5X T4 DNA ligase buffer (Invitrogen), 4 uL of 10 mM dNTPs mix, 2.5 uL of T4 DNA polymerase (3 U/uL), 1 uL of Klenow DNA Pol (5 U/uL) and 2.5 uL of T4 polynucleotide kinase (10 U/uL, NEB). After incubation for 30 min at 20C the DNA was purified using “QIAquick PCR Purification kit”. To add dA to the termini, 32 uL DNA from the prior step was mixed with 5 uL of 10X Klenow buffer, 10 uL of 1 mM dATP, and 3 ul of Klenow exo-polymerase (3’ to 5’ exo minus, 5 U/uL, NEB) and incubated for 30 min at 37C. DNA was purified using a “QIAquick MinElute Reaction Clean-up” (Qiagen) kit and eluted into 12 uL of water. To ligate Illumina adapters, 10 uL of cDNA from the prior step was mixed with 5 uL of 5X T4 DNA ligase buffer, 6 uL of adapter oligo mix, 4 uL of T4 DNA ligase (NEB) and incubated for 15 min at 25C. DNA was purified using a “QIAquick MinElute PCR Purification Kit”. cDNA was size-fractionated (typically with average fragment length of 170 base pairs) on 3.5% (w/v) “NuSieve GTG” agarose. The fractionated libraries were PCR amplified using “Phusion Hot Start High-Fidelity” DNA polymerase (NEB) and the following PCR protocol: 30 sec at 98C, then 10 sec at 98C, 30 sec. at 65C, and 30 sec. at 72C for 18 cycles, followed by a 10-min extension step at 72C. 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.