L285_TZ, T285_TZ in article
Total RNA extraction   Annual rings from TZ and SW were excised with a scalpel while keeping the increment core frozen. SW sample consisted of four to five pairs of annual rings located at approximately four or five annual rings away from the TZ. Samples were ground to fine powder in liquid nitrogen using first mortar and pestle and subsequently a milling machine (Qscillating Mill MM400 Retsch GmbH, Germany).       Total RNA isolation for SW and TZ samples was carried out as described by Chang et al.(1993) with minor modifications. After overnight lithium chloride precipitation, the RNA pellet was collected by centrifugation at 10 000 g for 30 minutes at 4°C, washed with 70% cold ethanol, air-dried for 3 minutes and dissolved in 50 µL nuclease-free Milli-Q water. Genomic DNA was digested with DNase I, followed by purification with RNeasy Plant Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. The concentration and the quality of the genomic DNA-free total RNA was assessed with spectrophotometer (GeneQuant 1300 Eppendof, Germany) and agarose gel electrophoresis.    Sample preparation for RNA sequencing  Eight micrograms of genomic DNA free total RNA was selectively depleted from ribosomal RNA (rRNA) according to the procedures described in RiboMinus™ Plant Kit for RNA-Seq (Invitrogen, USA). 500 ng of rRNA-depleted RNA used for transcriptome library preparation. After the ligation of SOLiD adaptor mix and reverse transcription, size fractionation of the transcriptome library was performed with gel size selection of 150-200bp cDNA, followed by amplification and purification according to the protocols described in SOLiD Total RNA-Seq Kit (Invitrogen, USA). Next, paired-end sequencing was conducted with SOLiD 5500xl at Institute of Biotechnology, University of Helsinki.