GSM2113579: MUT-IP-R3; Arabidopsis thaliana; RNA-Seq
Total RNA was extracted with modified CTAB method. Briefly, well ground materials were suspended in five volume of 65°C pre-heated extracting buffer (100 mM Tris-HCl pH 7.9, 2% CTAB, 2% PVP-30, 2 M NaCl, 25 mM EDTA, 0.05% spermidine, and fresh 2% β-ME). The mixture was heated at 65°C for 10 minutes, and extracted with chloroform/isoamyl alcohol (24:1) twice. The upper layer was precipitated with 1/2 volume 8 M LiCl for 2 hours to yield >200 nt RNA. Total RNA was treated with RNase-free DNase I (New England BioLab) in order to remove DNA contamination for reverse transcription, or directly used for mRNA isolation. Poly(A) RNA was isolated from total RNA using the Dynabeads Oligo dT (Invitrogen). The mRNA was qualified by Agilent 2100 Bioanalyzer. RNA was randomly fragmented to ~100 nt by RNA Fragmentation Reagents (New England Biolab), and incubated with 5 μg m6A-specific antibody (Synaptic Systems, Cat no. 202003 Lot no. 202003/53) overnight at 4 °C. The m6A-containing fragments were pulled down with BSA pre-blocked Dynabeads Protein A (invitrogen), and eluted with 6.7mM m6A-containing buffer. Eluted RNA was precipitated by 75% ethanol. NEBNext Ultra RNA Library Prep Kit (E7530) was used for library building on input and IP sample.
GEO Accession
GSM2113579