title: Illumina HiSeq 4000 paired end sequencing; Cellular and gene expression patterns associated with root bifurcation in Selaginella
library_construction_protocol: To identify possible molecular mechanisms with a role in root branching initiation in S. moellendorffii, we employed the developmental root branching assay for an RNA-seq experiment and sampled root tips on each day from 0 to 5 d after the first branching (time point 0), in which 300 µm apical parts were sampled to enrich for the meristematic region, while non-meristematic root regions were sampled separately. Sampled roots in the time course (300 root tips per sample, 4 biological repeats per time point) were cut off using a pair of stainless microscissors and then stored in RNAlater® RNA Stabilization Solution (Thermo Fisher Scientific) according to the manufacturer's instruction. Subsequently, microdissection was performed to collect root apices of 0.3 mm long, as well as non-meristematic regions between apices and branching point for the 5 d post first branching samples. Plants were grown aseptically under a 16-h light/8-h dark photoperiod at 20.25–43.2 μmol/m2/s in a 24°C growth chamber and roots developed after the transfer of explants to fresh MS1/2 medium. For RNA extraction, samples were ground three times using a QIAGEN Retsch Tissuelyser (30 Hz, 30 s). RNA extraction was done via TRIzol™ reagent (Thermo Fisher Scientific) and the RNeasy Plant Mini Kit (Qiagen) according to the provided protocols. Per sample, an amount of 250 ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version: Part # 15031047 Rev. E - October 2013) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA PolymeraseI and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally, enrichment PCR was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library.