GSM3640103: RNAseq_RNaseR_seedling; Oryza sativa; RNA-Seq
Briefly,the purified nuclei were used for extraction of high quality genomic DNA using three rounds of phenol, phenol:chloroform, and chloroform extraction coupling with ethanol precipitation. The purified genomic DNA was fragmented into main sizes between 200-500bp using sonication. The 5μg sonicated DNA with or without RNase H treatment was purified using Qiaquick PCR purification kit and incubated with 30μl of well characterized S9.6 antibody bound Dynalbeads overnight at 4°C in IP buffer (10mM NaPO4, 140mM NaCl,0.05%Triton X-100). After washing with IP buffer three times, Dynalbeads was eluted with elution buffer (50mM Tris-HCl pH8.0, 10mM EDTA, 0.5% SDS) two times with 250μl at 65°C for 15 mins each. Antibody bound DNA was finally purified using phenol-chloroform extraction and pelleted with ethanol. Air-dried DNA was resuspended in 20 μl of 10 mM Tris-HCl (pH 8.0). dDRIP-seq:Directional DRIP-seq libraries were prepared using DRIPed DNA above followed the published protocol with minor modifications(Wang et al. 2011). The library preparation directly started from the second strand synthesis step by skipping preparation of mRNA fragments and the first cDNA synthesis steps. The dUTP incorporated second-strand DNA was synthesized using DNA polymerase I. The remaining steps were exactly following the published protocol mentioned above. The final DRIP-seq library was QCed and sequenced using Illumina Hiseq2500 platform. NBS-seq:The non-denatured sonicated genomic DNA, the same as the one used in dDRIP experiment, was used for bisulfite treatment using EZ DNA methylation-gold kit purchased from ZYMO research. The bisulfite reaction and purification f bisulfite treated DNA was exactly followed the manual. The final purified DNA was used for BS-seq library preparation using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina purchased from NEB (Cat#: E7645S) RNAseq_RNaseR: Total RNAs were prepared using Trizol (Cat.# 15596026, ThermoFisher Scientific) followed by DNase I treatment and removal of majority of mRNAs with oligo(T)-conjugated magnetic beads purchased from Promega (Z5310). Approximately 80 μg RNAs were treated with RNase R (Cat.# RNR07250, Lucigen) one times followed by adding “A” base and treating with RNase R one more time. Finally, about 1μg RNAs were used for directional RNA-seq library preparation according to published procedures. The final RNase R-treated RNA-seq library was QCed and sequenced using Illumina X-ten platform.
GEO Accession
GSM3640103