GSM3660545: RIP-seq control rep1; Arabidopsis thaliana; RIP-Seq
Briefly, 2 g of crosslinked plant materials were ground into fine powder and suspended in Chromatin Extraction Buffer. The nuclear pellets were collected by centrifugation and washed for 4-5 times with NRBT Buffer. The pellet was resuspended in 150 μL Lysis Buffer and sonicated for 10 minutes. The supernatant was diluted with ChIP Dilution Buffer. Anti-Myc antibody and protein-A beads were added and incubated for 3 hours. Then, the beads were collected and washed with RIP Washing Buffer for 3 times. The CLF-RNA complex was eluted by RIP Elution Buffer. After de-crosslinking, the CLF-associated RNA was purified by TRIzol. Libraries were prepared according to NEBNext Ultra II RNA Library Prep Kit. Briefly, the CLF-associated RNA was fragmented by alkaline hydrolysis. Then, the first-strand cDNA was synthesized by using Random primers. After the synthesis of the second-strand cDNA, the end-repaired reagents were used to yield a protruding 3- 'A' base for ligation of Illumina's adaptors. After ligation, the libraries were amplified and ~200 bp PCR products were collected for sequencing.
GEO Accession
GSM3660545