GSM9765599: 3HA-AGO1 IP Rep 4; Arabidopsis thaliana; RIP-Seq
Tissues were ground in liquid nitrogen with a mortar until a fine powder was obtained. The frozen powder was mixed with 1 mL 4°C IP buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% (v/v) Nonidet™ P40 Substitute, RNase Out (Invitrogen™, #10777019) and 1 tablet of cOmplete EDTA-free Protease Inhibitor Cocktail / 50 mL (Roche, #11873580001). Immunoprecipitation using HA-Trap Magnetic Agarose (ChromoTek, #atma) was then performed. Lysates were centrifuged at 10,000 × g, 4°C for 10 minutes and the supernatant passed through a cell-strainer (40 µm, Falcon, #352340). A 200 µL aliquot was reserved for the input fraction, and the remaining lysate was incubated with HA-Trap Magnetic Agarose (ChromoTek, catalog ID: "atma") while rotating end-over-end for 2 hours at 4°C.  Beads were washed 5 to 6 times with 4°C IP buffer. To elute bound HA-AGO complexes, beads were incubated at 95°C for 5 minutes in nuclease-free water with a dry bath. Total RNA was then extracted from both the input and IP fractions, followed by RT-qPCR analysis and sRNA-seq library construction. Small RNA libraries were made using the NEBNext Low-bias Small RNA Library Prep Kit (New England Biolabs E3420L) with NEBNext LV Unique Dual Index Primers (New England Biolabs E3400, E3402, E3404, E3406, and E3408). Amplified libraries were evaluated using an Agilent TapeStation device and D1000 Screen Tape. TapeStation-calculated concentrations were used to normalize library concentration during pooling. The pool of completed libraries was purified using a Blue Pippin device (Sage Biosciences) to retain DNA in the 145-195 bp size range and remove any remaining free adapters. The quality and size of the pooled libraries was then assessed with the DNA 5000 Screen Tape on the Agilent TapeStation 4150 (Agilent Technologies), and the concentration was determined via qPCR using the KAPA Library Quantification Kit for Illumina (KAPA Biosystems). The libraries were then sequenced in single end mode on the Illumina NextSeq 2000 using a P4 50 cycle kit (Illumina) at 1 x 50 nucleotides. The pooled data were then de-multiplexed using unique dual indices to produce FASTQ files for each sample.