GSM6249084: Total mRNA fraction at 15 mins recovery rep 1; Arabidopsis thaliana; RNA-Seq
mRNA-sequencing: Frozen tissue was ground into a fine powder using a ⅛” steel ball bearing with 1 min shaking at 25 Hz in a TissueLyser II (Qiagen). Total RNA was extracted from finely ground tissue using TRI reagent at a ratio of 1 mL solution per 100 mg ground tissue (Sigma-Aldrich, #T9424-200ML). Residual phenol was removed from the crude extract through two chloroform extractions at a ratio of 1:5 v/v, followed by precipitation using isopropanol at 1:1 v/v. Precipitated RNA was washed twice with 70% ethanol and resuspended in a 1 mM sodium citrate buffer (pH 5.4). RNA quantification was performed through spectrophotometric analysis at 260 nm using the Nanodrop ND-1000 Spectrophotometer and RNA quality was assessed using 1% agarose gel electrophoresis or on the LabChip GX Touch (PerkinElmer). polysome profiling: Polysome-associated mRNA sequencing was performed using an adapted protocol (Lecampion et al. 2016). Briefly, 250 mg of frozen and ground plant tissue was dissolved in 1 mL of polysome extraction buffer [160 mM Tris-Cl (pH 7.6), 80 mM KCl, 5 mM MgCl2, 5.36 mM EGTA (pH 8), 0.5% IGEPAL CA-630, 40 U/mL RNasin Plus RNase inhibitor (Promega), 150 µg/mL cycloheximide, and 150 µg/mL chloramphenicol] and incubated on ice for 10 minutes. Samples were repeatedly centrifuged at 16,000 rcf for 5 minutes at 4°C until the supernatant was clear. Samples were loaded onto sucrose gradients, consisting of layers of 50% (1.68 mL), 35% (3.32 mL), 20% (3.32 mL) and 20% (1.68 mL), with the two 20% layers added separately (Lecampion et al. 2016). The buffer used in the gradients consisted of 400 mM Tris-Cl (pH 8.4), 200 mM KCl, 100 mM MgCl2, 10.12 µg/mL cycloheximide and 10.12 µg/mL chloramphenicol. Gradients were centrifuged at 41,000 rpm with an SW41Ti rotor for 2 hours at 4°C. Each gradient was passed through a spectrophotometer from highest to lowest density and absorbance at 260 nm recorded, with 1 mL fractions collected. Fractions were pooled into monosomal and polysomal sets and extracted using 5:1 acid phenol:chloroform. Briefy, an equal volume of the phenol:chloroform mix was added to each fraction, before centrifugation at 16,000 rcf for 10 minutes. The aqueous layer was extracted a second time using a one-fifth volume of chloroform, before the RNA was precipitated using an equal volume of isopropanol in addition to sodium acetate (pH 5.2) to a final concentration of 300 mM. The precipitated RNA was washed twice with 70% ethanol and resuspended in a 1 mM sodium citrate (pH 5.4) solution. Total RNA was extracted from finely ground tissue (as above) using TRI reagent at a ratio of 1 mL solution per 100 mg ground tissue (Sigma-Aldrich, #T9424-200ML). Residual phenol was removed from the crude extract through two chloroform extractions at a ratio of 1:5 v/v, followed by precipitation using isopropanol at 1:1 v/v. Precipitated RNA was washed twice with 70% ethanol and resuspended in a 1 mM sodium citrate buffer (pH 5.4). RNA quantification was performed through spectrophotometric analysis at 260 nm using the Nanodrop ND-1000 Spectrophotometer and RNA quality was assessed using 1% agarose gel electrophoresis or on the LabChip GX Touch (PerkinElmer). PolyA-enriched RNA-sequencing libraries were constructed using the Illumina TruSeq Stranded mRNA kit as per the manufacterer's instruction, except reaction volumes were scaled-down by 1/3.