NextSeq 500 sequencing; ChIP-seq of Oryza sativa ssp. indica epilines
ChIP-seq of Oryza sativa ssp. indica epilines
The sample material was 12 pooled fifth leaves of individual plants with two biological replicates for the control line and the epiline LR2, which belongs to the 5th selfing of the EUE selection pedigree.   LR2 and the control line were grown in soil in a growth chamber at 26 C / 21 C (day / night) for 24 days with a 16 h light/8 h dark regime (light intensity was 300 mol m-2 sec-1, the relative humidity was kept at 71%). Liquid Nitrogen Fixation was performed by means of pre-cooled (-80°C) mortar, pestle, metal spatula, and sample, which are placed on dry ice. Approximately 1 g per sample was grinded to a fine powder, put into the plant fixation buffer (40 ml Plant Cross-linking and Extraction Buffer, 1 ml 37 % formaldehyde, and 400 µl 0.1 M PMSF), mixed in vacuum for 10 min, 1.6 ml 2.5 M glycine added and mixed for 5 min in vacuum. The samples were transferred to a 50 ml conical tube and spun for 10 min at 2,500 rpm at 4 °C. The pellet washed twice in 10 ml ice-cold diH2O, spun for 10 min at 2,500 rpm and 4 °C, and the pellets were frozen on dry ice. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. Chromatin was prepared by resuspending each pellet in 5 ml Nuclei isolation buffer, 5 µl protease inhibitor cocktail (PIC), and 50 µl 0.1M PMSF. Samples were incubated on ice for 10 min, homogenized with 20 strokes of bouncing, centrifuged for 10 min at 2,500 rpm and 4 °C, and each pellet was resuspended in 500 µl DOC sonication buffer (5 µL PIC and 5 µl 0.1 M PMSF), incubated on ice for 10 min, and 50 mg of glass beads were added. The samples were sonicated twice with a microtip, 10 min in total, at 30 s pulse and 30 s pause, amplitude at 45 %. The tubes were centrifuged to pellet debris at maximum speed for 10 min at 4 °C. 10 µl supernatant was distributed for two replicates for Input DNA preparations.   The assays for histone modification H3K4me3- and PolII-detection were performed and provided by Active Motif, Inc. Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. After a final PCR amplification step, the resulting DNA libraries were quantified.
Experimental Factor: immunoprecipitate
anti-PolII
Experimental Factor: genotype
LR2