title: NextSeq 500 sequencing; Rice Lateral Root Inducible System timeseries
design: Rice Lateral Root Inducible System timeseries
library_construction_protocol: root sections between 750 µm and 2000 µm from the root tip were microdissected Root parts (750 um - 2000 um from the root tip) were dissected with razor blades. Rice seeds were germinated in Petri dishes on three layers of wetted whatman paper saturated with a 5 µM N-1-naphthylphthalamic acid (NPA) watery solution for two days at 28°C and uniformly grown seedlings were transferred to 250 mL-Erlenmeyer flasks containing 50 mL of liquid half strength Murashige and Skoog medium (MS1/2) supplemented with 5 µM NPA. Flasks were put on a shaker (120 rpm) in a growth room at 24 °C, 55 μE/m−2 s−1 light intensity, and 16 h/8 h day/night regime for 3 days. For treatment, the medium in the flasks was replaced by  MS1/2 supplemented with 5 µM 1-naphthalene acetic acid (NAA) or NPA as a control. 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 indexing adapters were ligated introducing single distinct 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. total RNA was extracted using  Trizol (Invitrogen) and the RNeasy Plant mini kit (Qiagen) 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. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which indexing adapters were ligated introducing single distinct 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.