GSM8942650: Tension_Bio1_Region1; Populus trichocarpa; RNA-Seq
The debarked stem segments of normal and tension wood were frozen with liquid nitrogen and stored at -80℃. The segments were mounted vertically onto cryostat chucks with Tissue-Tek O.C.T. Compound (Sakura Finetek) and sectioned into 16-μm slices using a cryostat (Leica, CM1900) at −20℃. The sectioned samples were placed on PET membranes of metal-frame slides and dehydrated by 99.5% ethanol. Laser capture microdissection (LCM) technology was applied to collect normal or tension xylem samples at different differentiation stages on debarked stem cross-sections. Five regions were cut by laser capture microdissection (Leica, LMD7000), and Region 1, 2 and 3 were collected (Figures 5A and 5B). The width of each region was set to three times the average width of 10 consecutive libriform fibers. Approximately 1,600,000 μm2 of each region of samples were collected and pooled as one biological replicate. Three biological replicates were collected for normal and tension xylem. After laser dissection, the collected regions of samples fell into 500-μL tube caps containing 50 μL of RLT buffer (QIAGEN) with 1% beta-mercaptoethanol (Sigma-Aldrich), which was then frozen with liquid nitrogen within 30 min and stored at -80℃. Total RNA was extracted using RNeasy Plant Mini Kit (QIAGEN) following the manufacturer instructions. The sequencing library was constructed using QIAseq® UPX 3' Transcriptome Kit (QIAGEN) according to manufacturer's guidelines.