title: GSM4661072: Riboseq_12hr_Rep1; Zea mays; OTHER
library_construction_protocol: For RPF generation, the remaining lysate was adjusted to A254 = ~10  (Lysate - PEB) with PEB. 600 ul of the adjusted lysate were digested with 15 ul RNase1 (Ambion AM2295) for 30 min at 28 ˚C on a shaker with slow speed. To terminate RNase digestion, 15 ul superase-IN (Ambion AM2696) were added. For each sample, two-aliquots of 600 ul (technical reps) were used and pooled after ultracentrifugation. The RNAse-digested lysate was layered on a precooled 330 ul sucrose cushion (1M sucrose, 40 mM Tris-acetate (pH 8), 100 mM KCl, 15 mM MgCl2, 100 ug/mL cycloheximide, 100 ug/mL chloramphenicol, 10 mM B-mercaptoethanol) in ultracentrifuge tubes (ThermoScientific #45237) and subjected to ultracentrifugation at 131k g (57000 rpm) for 90 minutes at 4˚ C in a Sorvall mini ultracentrifuge (Discovery M150) with S150-AT fixed angle rotor (ThermoScientific 45582). The pellet was resuspended in 150 uL ribosome dissociation buffer (10 mM Tris-HCl (pH 7.5), 10 mM EDTA (pH 8), 5 mM EGTA (pH 8), 100 mM NaCl).   RNA was purified by Trizol extraction method until the phase separations step followed by addition of an equal volume of ice cold 100% ethanol to the aqueous phase and further purification using Zymo RNA Clean & ConcentratorTM-5 columns (Zymo R1016) according to manufacturer's protocol and quantified using a Nanodrop spectrophotometer. Quality of RPFs was assessed by electrophoresis of denatured RNA in a 15% TBE-Urea gel (Invitrogen EC6885BOX) at 120V for 5 minutes and 200V for 55 minutes, staining the gel with SYBR gold (Invitrogen S11494) and determining the sharpness of the RPF band between the 28 nt and 34 nt RNA size markers. If the RPF bands appeared sharp for all samples, the RNAs from two technical duplicates were pooled for each sample. Subsequently, 10 ug RNA were treated with 1 uL Turbo DNase (Ambion AM2238) in a 50 uL reaction with 1x Turbo DNase buffer at 37 ˚C for 30 min followed by addition of 1 uL more Turbo DNase and incubation at 37 ˚C for additional 30 min, purification by Zymo RNA clean & concentrator -5 columns (Zymo R1016) and quantification with a Nanodrop spectrophotometer. Quality of the DNase-treated RNA was assessed as above. rRNA depletion was carried out using a half reaction of Ribo-Zero for plant seed/root kit (Illumina MRZSR116) per ~5-10 ug of DNAse treated RNA sample followed by RNA clean up using Zymo RNA clean & concentrator -5 columns (Zymo R1016) according to the Illumina TruSeq Ribo Profile kit protocol (Illumina 15066016). RNA was quantified using a Qubit RNA HS kit (Invitrogen Q32852) yielding ~20% recovery from the input.   For size-selecting the RPFs, denatured rRNA-depleted RNA was subjected to electrophoresis on a 15% TBE-Urea gel (Invitrogen EC6885BOX) at 120V for 5 min and 170V for 85 min, stained in the gel with SYBR gold (Invitrogen S11494), and visualized on a blue light transilluminator.  The gel region between 28 nt and 34 nt RNA size markers was excised and transferred to a 0.5 ml tube with a hole at the bottom made by a 18 G syringe needle, and the tube was placed a 2 mL microfuge tube.  The tube was then centrifuged at 21k g for 2 min at 4˚ C to crush the gel slice and transfer its contents to the 2 mL microfuge tube. 500 uL RNA gel extraction buffer (0.3 M NaOAc (pH5.5), 1 mM EDTA (pH 8), 10 mM Tris-HCl (pH 7.5), 0.25% (w/v) SDS) was added and incubated overnight at 4 ˚C on a shaker. Eluted RPFs were filtered through 0.22 µ SpinX cellulose acetate filter columns (Sigma-Aldrich CL8161). 2 uL Glyco Blue (Ambion AM9516) and equal volume of ice cold 100% isopropanol were added to the supernatant, and RPFs were precipitated at -80 ˚C for 3 hours followed by centrifugation for 45 min at 21k g at 4 ˚C, washing with ice cold 80% ethanol and resuspending the pellet in 3.5 uL water.  Prior to library preparation, RPF ends were repaired as follows using T4PNK kit (ThermoScientific EK0031): incubate 3.25 uL RNA at 70˚ C for 3 min, transfer to ice and centrifuge, add 0.5 uL 10 x T4 PNK buffer A (no ATP), 0.25 uL superase-IN, 0.5 uL T4PNK enzyme, incubate at 37 ˚C for 30 min, add 0.5 uL 10 mM ATP, incubate at 37 ˚C for 1 hr, add 5.5 uL water, deactivate the reaction by incubating at 75 ˚C for 10 min and transfer to ice. 250 mg of 10-day old maize B73 seedling roots (in biological duplicates) were ground with liquid N2 in 2.5 mL of polysome extraction buffer (PEB; 50 mM Tris-Acetate (pH 8), 200 mM KCl, 15 mM MgCl2, 1% (v/v) Triton X-100, 2% (v/v) polyoxyethelene 10-tridecylether, 200 mM sucrose, 100 g/mL cycloheximide, 100 g/mL chloramphenicol, 20 mM -mercaptoethanol). The crude lysate was filtered through a 40 µm cell strainer (Falcon 08-771-1) by centrifugation at 4000 g for 2 minutes. The flow through supernatant was further clarified by centrifugation at 21k g for 15 min. 300 l of clarified lysate were aliquoted for total RNA extraction.  Subsequent steps for ribosome profiling and RNAseq are described above in SAMPLES section cDNA libraries were prepared using Nextflex small RNA-Seq kit v3 (Bioo Scientific 5132-05) according to manufacturer's protocol and 12 cycles of PCR. Quality of the libraries was assessed using an Agilent bioanalyzer high sensitivity DNA Assay kit. Libraries were quantified using Qubit dsDNA HS Assay kit (Invitrogen Q32854) and diluted to same concentration. The riboseq and RNAseq libraries were pooled together for multiplexing and sequenced in two lanes using an Illumina HiSeq 3000 to yield single-end 50 bp reads. Because of low depth of riboseq sequences, riboseq samples were pooled and resequenced in additional two lanes using Illumina HiSeq 3000 to yield single-end 50 bp reads. Ribo-seq and RNA-seq
attributes.tag: GEO Accession
attributes.value: GSM4661072