experiment_id1061
Illumina sequencing of Brösarp-11-135 genomic fragment library
For genomic DNA library preparation we use young Arabidopsis seedlings, 5-8 plants for each accession. DNA was extracted from roots, which were ground to a fine powder in a liquid nitrogen and mixed with 2XCTAB DNA extraction buffer (100 mM Tris-HC1 pH 8, 1.4 M NaCl, 20 mM EDTA pH 8.0, 2% CTAB). After incubation at 65C for 30 min, DNA was extracted with equal volume of Chloroform and precipitated with 0.8 volume of isopropanol. Libraries were prepared using slightly modified Illumina Genomic DNA Sample Prep protocol. Briefly, DNA was fragmented by sonication with Bioruptor (Di-agenode); the peak of fragment sizes was about 400bp. End-repair of sheared DNA fragments, A-tailing and adapter ligation were done with NEBNext DNA Sample Prep Reagent Set 1 (BioLabs). Adaptor-modified DNA was resolved on 2% low melt agarose (Peqlabs) gel (including SybrGold nucleic acid gel strain, Invitrogen), run for 90 minutes at 100V. For size selection of the library DNA was excise from the gel with the size range 450bp. DNA was purified with a MinElute Gel Extraction Kit (Qiagen). The paired-end DNA libraries were amplified by PCR for 14-18 cycles with Illumina supplied PCR primers 1.1 and 1.2. Libraries were sequenced on Illumina GAII and HiSeq Analyzers using manufacturers standard cluster generation and sequencing protocols, with 76 and 100 bases read length.
library_id1061
GMI-MPB
experiment_id1061