alias: GSM2466044
title: GSM2466044: W22-ref AGO18b 1st; Zea mays; RIP-Seq
library_construction_protocol: Frozen tissue was pulverized in liquid nitrogen by Grinding and was lysed in ice-cold lysis buffer (50 mM Tris 7.4, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 0.5% NP-40 and 0.5% deoxycholate) with freshly added 1mM DTT, 200 U/mL RNase inhibitor (Takara) and protease inhibitor cocktail (Roche) for 30 min on ice. After centrifugation for 30 min at 12000 rpm at 4 oC, the supernatant was transferred to a new tube and used for immunoprecipitation. For immunoprecipitation, 300 μL lysate was incubated with 10 μg antibody or control IgG antibody overnight at 4 oC. The immunuprecipitates were further incubated with protein A Dynabeads for 2h at 4 oC. After applying to magnet and removing the supernatants, the beads were sequentially washed with lysis buffer, high-salt buffer (250 mM Tris 7.4, 750 mM NaCl, 10 mM EDTA, 0.1% SDS, 0.5% NP-40 and 0.5 deoxycholate), and PNK buffer (50 mM Tris, 20 mM EGTA and 0.5% NP-40) for two times, respectively. The immunoprecipitates were eluted from the beads with elution buffer (50 nM Tris 8.0, 10 mM EDTA and 1% SDS) and the RNA was purified with Trizol reagent (Life Technologies, Invitrogen, Carlsbad, CA, USA). Carry the recovered RNA through a standard cDNA library preparation protocol originally described for cloning of small regulatory RNAs. The first step, 3' adapter ligation, was carried out as described on a 20 μl scale using 10.5 μl of the recovered RNA. Use the sequencing adapter sets described. Depending on the amount of RNA recovered, 5'-adapter-3'-adapter products without inserts may be detected after amplification of the cDNA as additional PCR bands. In such cases, excise the longer PCR product of expected size from a 3% NuSieve low-melting point agarose gel, elute the PCR product from the gel pieces using the GelElute kit (Qiagen).
identifiers.namespace: GEO
identifiers.id: GSM2466044