GSM2064502: Aspen rep2; Wolfiporia cocos; RNA-Seq
Extraction buffer was prepared by combining 10 ml 690 mM para-aminosalicylic acid (sodium salt) (Sigma-Aldrich, St. Louis, MO) with 10 ml 56 mM triisopropylnapthalene sulfonic acid (sodium salt) (Sigma-Aldrich), and placed on ice. To this was added 5 ml 5X RNB (1.0 M Tris, 1.25 M NaCl, 0.25 M EGTA). The pH of the 5X RNB was adjusted to 8.5 with NaOH. The mixture was kept on ice and shaken just before use. Frozen fungal pellets were ground to a fine powder with liquid nitrogen in an acid washed, pre-chilled mortar and pestle. The ground mycelia were transferred to Falcon 2059 tubes (VWR International, West Chester, PA), and extraction buffer was added to make a thick slurry. The samples were vortexed vigorously and placed on ice until all samples were processed.  One half volume TE-saturated phenol (Sigma-Aldrich) and ¼ volume chloroform (Sigma-Aldrich) were added to each sample and vortexed vigorously.  Samples were spun at 2940 x g in a fixed-angle rotor for 5 minutes.  The aqueous layer was removed to a new tube, and phenol:chloroform extractions were repeated until the interface between the aqueous and organic layers was clear. The final aqueous extractions were placed in clean 2059 tubes, to which was added 0.1 volume 3M sodium acetate, pH 5.2, (DEPC-treated) and 2 volumes absolute ethanol. The tubes were shaken vigorously and stored overnight at -20˚C.  The tubes were spun 1 hour at 2940 x g, the supernatants were decanted, and the pellets were resuspended in 4 ml RNase-free H2O. Total RNA was purified using the RNeasy Maxi kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol for RNA cleanup. RNAs were eluted from the RNeasy spin columns using two spins, for a final volume of 2 ml. The eluted RNAs were ethanol precipitated and stored overnight at -20˚C. Messenger RNA was purified from 1μg total RNA using poly-T oligo-attached magnetic beads. Double-stranded cDNAs were synthesized using SuperScript II (Invitrogen, Carlsbad, California, USA) and random primers for first strand cDNA synthesis followed by second strand synthesis using DNA polymerase I and RNAse H for removal of mRNA. Double-stranded cDNA was purified using Agencourt AMPure XP beads (Qiagen, Valencia, California, USA) as recommended in the TruSeq RNA Sample Prep Guide. cDNAs were end-repaired by T4 DNA polymerase and Klenow DNA polymerase and phosphorylated with T4 polynucleotide kinase. The blunt ended cDNA was purified using Agencourt AMPure XP beads. The cDNA products were incubated with Klenow DNA polymerase to add an ‘A’ base (Adenine) to the 3’ end of the blunt phosphorylated DNA fragments and then purified using Agencourt AMPure XP beads. DNA fragments were ligated to Illumina adapters having a single ‘T’ base (Thymine) overhang at their 3’end. The adapter-ligated products were purified using Agencourt AMPure XP beads. Adapter ligated DNA was amplified in a Linker Mediated PCR reaction (LM-PCR) for 15 cycles using PhusionTM DNA polymerase and Illumina's PE genomic DNA primer set and then purified using Agencourt AMPure XP beads. Quality and quantity of the finished libraries were assessed using an Agilent DNA1000 series chip assay (Agilent Technologies, Santa Clara, CA) and Invitrogen Qubit HS Kit (Invitrogen, Carlsbad, California, USA), respectively, and libraries standardized to 2 μM.
GEO Accession
GSM2064502