title: GSM1701508: 40h rep2 barcode1; Phanerochaete chrysosporium; RNA-Seq
library_construction_protocol: RNA was extracted from fungal colonized wood sections prepared as described above. Samples, comprised of 20 sections each, were harvested after 40h (4 samples) and 96 hr (4 samples), immersed in liquid nitrogen and stored at -80C until extraction. For total RNA purification, wood sections were ground in prechilled mortar and pestle in liquid nitrogen. Autoclaved and prechilled glass beads (0.5mm) were used during the grinding to make the wood sections into fine powder. Following repeated phenol;chloroform extractions, RNA was ethanol precipitated at -20°C overnight and further purified using the RNeasy mini kit (Qiagen, Valencia, CA). Total RNA was DNAase treated according to the manufacture’s protocol. 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.
identifiers.namespace: GEO
identifiers.id: GSM1701508