Illumina HiSeq 2000 sequencing; Study of changes in chromosomal interaction patterns of Flowering Locus C gene upon vernalization
Study of changes in chromosomal interaction patterns of Flowering Locus C gene upon vernalization
Whole seedlings were harvested. 2-3 g Col-Sf2 seedling tissue was collected. Bleach sterilized seeds were sown on 1xMS agar plates and stratified for 48 h at 4 °C. Seedlings were grown at 21 °C and 16 h long day conditions.  For vernalization, seedlings after 4 weeks of cold treatment were transferred back to 21 °C long day conditions for 7 more days.  Nuclei were isolated, resuspended in 250 µl 1.2 x buffer B (Roche) and treated with 0.2% (w/v) SDS.  3C analysis was performed similar as previously described with adaptations to A. thaliana  (Hövel et al., 2012; Louwers et al., 2009b) using BglII. To obtain 4C samples, DNA was digested with 50 U NlaIII (NEB, 5U/µl) and re-ligated for circularization in a 2 ml volume. 100 ng of re-ligated, precipitated DNA served as template in each inverse PCR to produce 4C libraries.