GSM3263634: RNA sequencing from RIP input 2; Arabidopsis thaliana; RIP-Seq
Plantlets were collected, frozen in liquid nitrogen and grind.NSRa protein tagged with HA was immunoprecipitated from the nrsa mutant background expressing the pNSRa:NSRa-HA  construct (Bardou et al., 2014). Briefly, 10 day old seedlings treated with 100 nM NAA for 24 hours were irradiated three times with UV using a UV crosslinker CL-508 (Uvitec) at 0.400 J/cm2. Plants were ground in liquid nitrogen and RNA-IP was performed as in Sorenson and Bailey-Serres (Sorenson and Bailey-Serres, 2014) with the following modification : immunoprecipitation (IP) was performed in duplicate using anti mouse HA-7 monoclonal antibody (Sigma) and the negative IP (Mock) was done using anti mouse IgG (Millipore). RNA was eluted from the beads with 50U proteinase K (RNase grade, Invitrogen) in 2 μl of RNase inhibitor at 55°C for 1 hr in wash buffer and extracted using Trizol according to manufacturer instructions. A tenth of the input fraction was saved for RNA extraction. RNA was extracted using TRIZOL according to manufacturer instructions RNA were depleted of rRNA using the plant leaf ribozero kit (Illumina) and libraries were prepared using the Illumina TruSeq Stranded mRNA library prep kit according to the manufacturer instruction but omitting the polyA RNA purification step and sequenced on a NextSeq500 sequencer (Illumina) using single-end 75 bp reads mode.