title: Comparison of wild type and DEK1 mutant juvenile gametophytic development in Physcomitrium (Physcomitrella) patens with RNAseq data
design: Comparison of wild type and DEK1 mutant juvenile gametophytic development in Physcomitrium (Physcomitrella) patens with RNAseq data
library_construction_protocol: Material for RNA extraction was harvested after 3, 5, 9, 12, and 14 days of growth, always at the same time of the day. The samples were frozen in liquid nitrogen and stored at -80°C until further processing. Three starting cultures of each strain were used to initiate parallel cultures (biological replicates) used for RNA extraction. Phenotypic characterization of the plant material has been performed using light microscopy and image analysis using ImageJ software. All five strains were grown in parallel in the same cultivation room. Starting cultures of protonemata were maintained on minimal media (BCD) at 16-h light [70–80 mmol m-2 s-1]/8-h dark at 25°C. Total RNA was extracted from frozen material using the RNeasy lipid tissue mini kit (Qiagen) with few modifications. Briefly, the frozen tissue was thoroughly homogenized using a tissuelyser with pre-frozen blocks. Approximately 120 μg of powdered tissue was lysed in 1 mL of QIAzol lysis reagent. Two hundred μL of chloroform was added and the mixture was centrifuged at 4°C. The aqueous phase was collected, 1.5 volumes of 100% (v/v) ethanol was added, and the mixture was vortexed. After binding of the RNA to the RNeasy mini spin column, on-column DNase I treatment was performed to remove genomic DNA. The column was washed with the RPE buffer (Qiagen), dried, and RNA eluted in 45 μL of ribonuclease-free water. The concentration of RNA was measured and RNA integrity was further assessed using an Agilent 2100 Bioanalyzer (DE54704553; Agilent Technologies) with an RNA 6000 LabChip kit. The RNA samples were stored at -80°C until sent for sequencing. Strand-specific TruSeqTM RNAseq library construction of 74 libraries was performed at The Norwegian High Throughput Sequencing Centre in Oslo.