NextSeq 500 sequencing; Hybrid aspen (Populus tremula x tremuloides) cambial zone RNA-sequencing with 35um wide fraction scheme
Hybrid aspen (Populus tremula x tremuloides) cambial zone RNA-sequencing with 35um wide fraction scheme
35 μm cryo-fractions were collected across the cambial zone from three 3-month old WT (T89) and pLMX5::AtIPT7 line 1 and 3 trees (around 20th internode). Tissue identity of cryofractions was confirmed through safranin-alcian blue stained cross-sections. Gene expression profiles were analyzed in twelve 35 μm cryosections collected across the cambial zone of three WT and two pLMX5::AtIPT7 line 1 and 3 trees. P. tremula × tremuloides clone T89 was transformed with the LMX5::AtIPT7 construct. ARABIDOPSIS THALIANA ISOPENTENYLTRANSFERASE 7 gene (AT3G23630) was cloned into a Gateway vector with 1807 bp LMX5 promoter from P. tremula × tremuloides Transformation was done by agrobacterium. Clone trees were propagated from in-vitro cultivation. Total RNA was extracted from cryosections using the RNeasy plant mini kit (Qiagen) with ‘‘RLT’’ lysis buffer and an RNase-free DNase set (Qiagen) to remove any remaining genomic DNA. An Ovation Universal RNA-Seq System kit was used for Illumina library preparations (NuGEN Technologies Inc., CA, USA). Purified total RNA (10-50 ng) was used and primers for ribosomal removal were designed and used as outlined in the kit manual. Libraries were purified with AMPure XP beads (Beckman Coulter Inc., MA, USA) and quantified.
Experimental Factor: WT
genotype