GSM4629460: Col input_Rep3_sRNA; Arabidopsis thaliana; ncRNA-Seq
Total RNAs were isolated from 15-day-old seedlings with TRI reagent (Molecular Research Center, TR118). Total RNA extracted from 15-day-old seedlings of both wild type and mutants. For library construction, 30μg total RNA were resolved on a 15% polyacrylamide gel with 8M urea. Small RNAs in the 15-40 nt size range were recovered as described (Liu et al., 2012). Small RNA libraries were prepared using the library preparation kit (NEB, E7100), and sequenced with an Illumina HiSeq2500 platform at BerryGenomics, China. For AGO1 IP, 1g of 15-day-old seedlings were grinded in liquid nitrogen and IP buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10% Glycerol, 0.1% NP-40, 1x cocktail and 1x PMSF) was added with incubating for 20 min with gently shaking at 4°C. Before immunoprecipitation, the supernatant was incubated with 100μL of dynabeads (Invitrogen, 10002D) for 2h at 4°C. After centrifuge and filtered, the supernatant was used for IP. 50 ul was taken out as input, the rest were incubated with AGO1 antibody (Agrisera, AS09527) for 2 h at 4 ℃. The beads were washed with wash buffer (IP buffer with 0.5% NP-40), 1/10 volume was added 2x SDS-loading buffer for western blots and 9/10 volume was for RNA isolation. And the small RNA libraries were prepared using the library preparation kit (NEB, E7100), and sequenced with an illumina Nextseq platform at BerryGenomics, China.
GEO Accession
GSM4629460