title: Jefferson Bark Transcriptome Sequencing
design: Total RNA was isolated using a protocol previously described (Filichkin, 2007).  RNA was extracted using Plant RNA Reagent (Invitrogen).  Total RNA was treated for 10 minutes at 65 °C with RNAsecure reagent (Ambion). To eliminate genomic DNA contamination, all RNA preparations were treated with RNase-free Turbo DNase (Ambion) for 15 minutes at 37 °C. Total RNA was further purified using the RNeasy Mini RNA kit (Qiagen) according to the manufacturer’s cleanup 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 assessed using a ND-1000 spectrophotometer (Thermo Fisher Scientific) and Bioanalyzer 2100 (Agilent Technologies).      Fragmented RP or SMART-prepared 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 minutes at 20 ºC 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 minutes at 37 ºC. DNA was purified using a QIAquick MinElute Reaction Clean-up kit (Qiagen). 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 minutes at 25ºC.  DNA was purified using a QIAquick MinElute PCR Purification Kit. cDNA was size-fractionated (with average fragment length of between 325 to 350 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 seconds at 98 ºC, then 10 seconds at 98 ºC, 30 seconds at 65 ºC, and 30 seconds at 72 ºC for 18 cycles, followed by a 10-minute extension step at 72 ºC and purified using a QIAquick PCR Purification kit. Illumina cluster generation was done in the Oregon State University CGRB core facility using a standard Illumina protocol.
library_name: Jefferson-bark