GSM4273166: TOC1_DAP_rep1; Oryza sativa; OTHER
Genomic DNA was extracted from rice mature leaves using the DNeasy plant mini kit (QIAGEN, 69104) according to the manufacturer's instructions. One micrograms of genomic DNA were diluted in EB (10 mM Tris-HCl, pH 8.5) and sonicated to 200-bp fragments in a Bioruptor (Diagenode). Purified samples were then end-repaired, and ligated with an adaptor using the NEBNext® Ultra™ II DNA library prep kit (New England BioLabs, E7645) according to the manufacturer's recommendations. DNA libraries were purified by bead cleaning using a 1:1 bead:DNA ratio, eluted from the beads in 30 μl of EB, and quantified with the Qubit HS fluorometric assay. Fifty microliters of purified Halo-OsTOC1 protein (2 μg plasmids per reaction) was diluted in 400 μl of 1X phosphate-buffered saline (PBS) containing 25 μl of washed Magne®HaloTag® beads (Promega) for 1 h at room temperature on a rotator to encourage the protein binding to the beads. In addition to the Halo-OsTOC1 samples, a negative control Halo sample was run simultaneously using protein expressed in the TNT wheatgerm expression system (Promega). The beads were washed four times in 1X PBS + NP40 (0.005%) and resuspended in 100 μl of 1X PBS. Fifty nanograms of the genomic DNA library was diluted to a final volume of 60 μl in 1X PBS and added to the protein-bound beads. Protein-bound beads and gDNA were rotated for 1 h at room temperature. The beads were washed four times in 1X PBS + NP40, followed by two washes with 1X PBS, transferred to a new tube, and DNA was recovered by resuspending in 25 μl EB and boiling for 10 min at 98 °C. Eluted samples were enriched and tagged with dual indexed multiplexing barcodes by performing six cycles of PCR in a 50-μl reaction. Samples were pooled and sequenced using an Illumina HiSeq X Ten system (paired-end 150-bp reads).
GEO Accession
GSM4273166