Illumina HiSeq 2000 sequencing; Ribo-Seq analysis of Col-0, nrf1-1 and nrf1-2 mutant under Fe sufficient and Fe deficiency condition
Ribo-Seq analysis of Col-0, nrf1-1 and nrf1-2 mutant under Fe sufficient and Fe deficiency condition
10d old 1/2 MS grown plants transferred to +Fe and -Fe for 2 days and then roots were harvested for polysome isolation. 10d old 1/2 MS grown plants transferred to +Fe and -Fe for 2 days and then roots were harvested for polysome isolation. 10d old 1/2 MS grown plants transferred to +Fe and -Fe for 2 days  mRNA from polysome were extracted with polysome extraction buffer B. Further sucrose gradient was used to isolate the polysome-mRNA complex. Further RNaseI treatment was done to digest the free RNA and ribose boundmRNA was extracted as ribosome protected fragments (RPF). Further purification of RNA was done and send for sequencing. the nucleic acid library was constructed by following the protocol described in Liu et al., 2013, Plant Cell and Ingola et al., 2012, Nature protocol.