title: GSM8193442: Leaf primordia SR1, Halo-tagged AmMIXTA, replicate 3; Nicotiana tabacum; OTHER
library_construction_protocol: Leaf primordia harvested from three independent WT plants were flash-frozen, grinded and DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega; A1120). DNA was fragmented using a sonicating bath for 20 min (Elam S10H Elmasonic) and fragment size was verified on gel. the DNA fragments were subjected to end-repair (Fast DNA end repair kit, Thermoscientific, K0771), A-tail reaction using Klenow enzyme (NEB, M0212S), ligation to the adaptor (Promega, M1794) and DNA concentration measurement (Qubit dsDNA Assay kit, Qiagen, Q32850). HALO-AmMIXTA, HALO-NtZFP8 and HALO-nlsGFP (negative control) were expressed independently three times using the TnT® Coupled Wheat Germ Extract System (Promega, L41030). Expression was confirmed by Western-blot using Monoclonal Antibody Raised Against the HaloTag® Protein (Promega, G9211) and Goat anti-mouse IgG (H+L) HRP conjugated (Chemicon, AP308P).  Proteins were bound to the 20μl of Magne® HaloTag Beads (Promega, G7882) per sample, washed and put in contact with 1000 ng of DNA for 1h at 25°C. Each replicate of proteins was put in contact with one replicate of DNA. Beads were washed and DNA was eluted in EB at 98°C. Index and adaptor were added to the DNA fragments using Phusion® High-Fidelity DNA polymerase (NEB, M0530L) and NEBNext® Multiplex Oligos for Illumina® primers (NEB, E6440S), and DNA check on gel and was purified using the minelute reaction cleanup kit (Qiagen, 28204)