Ribosome-Protected Footprint Isolation and Ribo-seq Library Construction. Pulverized frozen root tissue (2.5 mL) was thawed in 5 mL of PEB and a S16 fraction was obtained as briefly described above and detailed by Mustroph et al. 2009 PNAS. Polysomes were isolated by TRAP and digested with RNAse. EZview Red anti-FLAG M2 magnetic beads (250 μL) were prewashed twice with RNAse Digestion Buffer [RNID; 20 mM Tris·HCl (pH 8.0), 140 mM KCl, 35 mM MgCl2, 50 μg/mL cycloheximide, 50 μg/mL chloramphenicol]. These were added to the S16 fraction and binding of FLAG epitope-tagged ribosomes associated with RNA was achieved by by incubation with gentle rocking for 2 h at 4 °C. The supernatant was removed with a magnet and the beads were washed four times by resuspension in 5 mL RNID followed by capture. Ribosome footprint fragments (RFs) were obtained by resuspension of the beads in 1 mL of RNID buffer additionally containing 500 units of RNase I (Ambion; ca. 15 U/µg RNA) and incubation for 90 min at 23-25 °C with rocking. RFs were extracted from ribosomes with TRIzol. For fragment size selection, the RNA was resuspended in 10 µl of 10 mM Tris (pH 7.0) and separated on a 7 M urea-denaturing  polyacrylamide gel electrophoresis (PAGE) in a 15% polyacrylamide gel. Marker oligos were used to demarcate fragments of 26-32 nt, which were collected. Gel slices were processed in 250 µl gel extraction buffer [20 mM Tris (pH 7.0), 250 mM NaOAc, 1 mM; EDTA, 0.25% (w/v); SDS] by freezing at -80°C for at least 30 min and then incubation for 15 min at 23-25 °C with rocking. The eluate was recovered by centrifugation for 10 min at 13,000 x g and the RFs recovered by ethanol precipitation. RFs were dephosphorylated using T4 polynucleotide kinase, resuspended in 25 µl 10 mM Tris (pH 8.0) and denatured for 2 min at 75°C, equilibrated to 37°C and brought to a volume of 50 µl in 1 T4 polynucleotide kinase reaction buffer with 25 U T4 polynucleotide kinase (NEB M0201S) and 12.5 U SuperaseIn. This was incubated 1 h at 37°C and heat-inactivated 10 min at 70°C. Dephosphorylated RFs were ethanol precipitation and linkers were ligated for 2.5 hr at 37°C in a 20 µl reaction consisting of the RFs, 12.5% w/v PEG 8000, 10% DMSO, 1x T4 Rnl2(tr) reaction buffer, 20 U SuperaseIn, 500 ng preadenylylated miRNA cloning linker (IDT, miRNA cloning linker #1), and 200 U T4 Rnl2(tr) (NEB, M0242L). Ligation products were separated by a denaturing 15% PAGE, the ligated-RFs were excised, recovered and resuspended in 10.0 µl of 10 mM Tris (pH 8). Reverse transcription was then performed in a 20 µL reaction by addition of 4 µL of 5× first-strand buffer, 1.0 μL of SUPERase•In, 1.0 μL of 100 mM DTT, and 1.0 μL of SuperScript III (Invitrogen) and incubation for 30 min at 48 °C. The reaction was heat- inactivated for 15 min at 98 °C, separated on a 10% (wt/vol) 7 M urea-denaturing PAGE in 0.5× TBE and stained with SYBR Gold. The 130 nt fragment region was excised and recovered. The cDNA was resuspended in 15 μL of RNase-free water and subjected to ssDNA circularization. The 20 μL reaction containing 1× CircLigase buffer, 1 μL of 1 mM ATP, 1 μL of 50 mM MnCl2 and 1 μL (100 U) CircLigase(Epicentre) was incubated at 60 °C for 1 h and heat-inactivated at 80 °C for 10 min. Contaminating rRNA fragments were partially removed by subtractive hybridization with biotinylated oligos corresponding to rRNA fragments abundant in RF libraries (101). Briefly, 5 μL of the circularization reaction was combined with 1 μL of 20× SSC, 1 μL of 200 μM subtraction pool and 3 μL of water; denatured at 100 °C for 90 s; and annealed to 37 °C. The biotinylated oligo/rRNA hybrids were recovered with Dynabeads MyOne Streptavidin C1 (Invitrogen) using 0.25 mg beads per sample according to the manufacturer’s instructions. The RFs of the subtracted libraries were precipitated and resuspended in 20 μL of water.  PCR amplification of libraries was performed in an 50 μL reaction of 1× Phusion HF buffer (New England Biolabs), 1 μL of 10 mM (each) dNTPs, 0.5 μL of 50 mM (each) library primers and indexing primers, 0.5 μL of Phusion HF polymerase (New England Biolabs), and 5 μL of rRNA-subtracted circularized template. Amplification was as follows: 30 s at 98°C and 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C. PCR products were separated on a 8% (wt/vol) PAGE using 1× Tris-Borate-EDTA. The amplified RF library (∼120-125 bp) was excised and recovered. Poly(A)+ RNA-seq libraries were prepared from 15 µg of DNase I (Thermo Scientific) treated RNA extracted from the S16 fraction as described by Wang et al., PlosOne 2011
GEO Accession
GSM2601708