NextSeq 500 sequencing; RNA-seq GLV6 overexpression during lateral root initiation in Arabidopsis thaliana
RNA-seq GLV6 overexpression during lateral root initiation in Arabidopsis thaliana
Collection of gravistimulation induced root bends from lab grown, 4-day old, Arabidopsis seedlings.  For each sample, gravistimulation induced root bends with a length of 2-3mm were dissected with a scalpel from about 400 seedlings. Surface sterilized seeds were grown on sterile nylon meshes placed on top of standard ½ MS medium with 1% sucrose and 0.8% plant tissue culture agar. After 2 days of stratification at 4°C in the dark, the square petri plates were incubated vertically at 21°C in continuous light. After 4 days, seedlings were gravistimulated by turning the plates 90° counterclockwise to stimulate bending of the growing roots. 8h after gravistimulation, the nylon meshes to which the seedlings were attached were moved to ½ MS medium with 2µM estradiol or an equivalent volume of DMSO as mock treatment. 3h and 6h after treatment, root bends were collected in a centrifuge tube and frozen in liquid N2 Per sample, a 100ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit, 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. Total RNA was extracted using the Qiagen RNeasy Plant Mini Kit in combination with on-column DNAse treatment according to the manufacturer's instructions. RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). 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, an 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.