GSM4190229: Sample 2 MUTEp::AGO1DAH; Arabidopsis thaliana; RIP-Seq
RNA immunoprecipitation experiments were conducted as previously described (Carbonell, 2012) with a minor modification.  In brief, four grams of Arabidopsis leaves from 6 day-old seedlings were ground in liquid nitrogen and dissolved in 12-ml protein lysis buffer (50mM Tris-HCl, PH 7.4, 2.5mM MgCl2, 100mM KCl, 0.1% Nonident P-40, 0.5mM PMSF, proteinase inhibitor cocktails (EDTA-free protease inhibitor cocktail Tablet/50ml lysis buffer, Roche), 50 units/ml RNase inhibitor (Thermo Scientific)). Cell extracts were centrifuged two times at 12,000 rcf at 4℃ for 15 min to remove cell debris. The supernatant was incubated with binding control agarose beads (Chromotek, Cat. No. bab-20) without antibody for 30min at 4℃ to remove the non-specific binding, then incubated with anti-GFP agarose beads (Chromotek, Cat. No. gta-20) for 2hr at 4℃.  The beads were washed 3 times by protein lysis buffer at 4℃.  RNA was recovered by incubating the beads in same volume of proteinase K buffer (50mM Tris-HCl, PH 7.4, 25mM EDTA, 300 mM NaCl, 1.25% SDS, 1μg/ul proteinase K (Thermo Scientific, Cat. No. AM2546)) for 15min at 65℃ and purified using the Zymo micro RNA kit (Zymo Research, Cat. No. R1061). Small RNAs were quantified using the Agilent 2100 Bioanalyzer system (Aglient small RNA Kit, Cat. No. 5067-1548). Small RNAs were converted to cDNA libraries using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, MA, USA).  5'- and 3'-adapters were ligated to the purified small RNA, followed by reverse transcription using Protoscript II reverse transcriptase and incorporation of index tags by PCR. The resulted libraries were subjected to size selection (145–160 bp) using Sage Pippin Prep (Sage science, MA, USA) on a 3% agarose gel.  The cDNA libraries were the sequenced using the Illumina Hiseq 2500 system, generating 50 bp single-end reads.
GEO Accession
GSM4190229