GSM4933424: Mock depletion CuAAC-NAD-seq-3; Arabidopsis thaliana; RNA-Seq
Total RNAs were extracted from 12 days Col-0 seedlings by TRIzol reagent (Molecular Research Center) and treated with 1 unit/μg DNase I at 37 ℃ for 1 h. (Roche). After DNase I treatment, total RNAs were phenol/chloroform extracted and ethanol precipitated. Poly(A) RNAs were isolated from total RNAs using Oligo d(T)25 Magnetic Beads (New England Biolabs) following the manufacturer's instructions. RNA concentration was determined by Nanodrop spectrophotometers. 10 μg mRNAs were subjected to Anti-m7G antibody treatment to remove the m7G-RNAs (Samples without adding Anti-m7G antibody as the Mock depletion). mRNAs in supernatants were acid phenol/chloroform extracted, ethanol precipitated and redissolved in DEPC H2O. Finally, redissolved mRNAs were separated to three aliquots. One aliquot contained ~150 ng mRNAs was subjected to mRNA-seq library construction, the other two aliquots contained ~3 μg mRNAs in each were made up to 100 μg by adding E. coli tRNA and further performed CuAAC- and SPAAC-NAD reaction. After CuAAC- or SPAAC-NAD reaction, RNAs were incubated at 25 ℃ for 1 h with 25 μl magnetic MyOne Streptavidin C1 beads, RNAs were finally eluted from beads by incubating with 20 μl H2O at 80 ℃ for 5 min. NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7420, New England Biolabs) was used for subsequent mRNA-seq and NAD-seq libraries construction. Firstly, three RNAs batches were incubated with RT mix in the kits containing First Strand Synthesis Reaction Buffer and random primers at 94 ℃ for 6 min to fragment mRNAs. The remaining steps were performed according to the manufacturer's instructions.
GEO
GSM4933424