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Department of Plant Sciences University of Cambridge

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Namespace:Department of Plant Sciences University of Cambridge

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Protocols: Chlamydomonas reinhardtii cells (CC-4350 cw Arg 7-8 mt+) (Chlamydomonas Resource Center [http://chlamycollection.org/strains/]) were maintained in 750 ml Tris-acetate-phosphate medium (Harris, 1989) at 23 C on a rotatory shaker (140 rpm) under constant illumination with white light (70 uE.m2.sec-1) to mid-log phase (OD750 ~ 0.6). Chlamydomonas cultures were harvested by filtering off the media, the cell paste was flash frozen and pulverized in liquid nitrogen with 5 ml of pre-frozen lysis buffer (20 mM Tris-Cl pH7.5, 140 mM KCl, 5 mM MgCl2, 100 ug/ml cycloheximide, 100 ug/ml chloramphenicol, 0.05 mM DTT, 0.1% NP40 and 5% sucrose). The frozen powder was thawed on ice and clarified by centrifugation for 30 min at 4700 rpm at 4 C followed by adjustment of A254 to ~4 before snap-freezing in liquid nitrogen and storage at -80 C. For Chlamydomonas, lysates were slowly thawed on ice and a 200 ul aliquot (A254 = 4) treated with 300 U RNAse 1 (100 U/ul, Life Technologies cat. no. AM2294) in a thermo-mixer at 28 C, 400 rpm for 30 min. The tube was placed on ice, 2 ul of SUPERase-In RNase inhibitor (20 U/ml, Life Technologies) added and the reaction layered onto a 1 M sucrose cushion prepared in Chlamydomonas polysome buffer (20 mM Tris-HCl pH 7.5, 140 mM KCl, 5 mM MgCl2, 0.5 mM DTT, 100 ug/ml cycloheximide, 100 ug/ml chloramphenicol and 0.5 ug/ml SUPERase-In.). The cushion was ultracentrifuged at 38,000 rpm (5 hr, 4C) in a Beckman Sw41Ti rotor. 150 ul of each cell lysate was digested with proteinase K and cellular RNA extracted using acidic phenol:chloroform as above. Ribosomal RNA was depleted from 5 ug of total RNA using a Ribo-Zero rRNA removal kit targeting the appropriate species following the manufacturers instructions (Human/Mouse/Rat: Epicentre, cat. no. RZH1046, Plant Seed/root: Epicentre, Cat. No. MRZSR116). Depleted RNA was resuspended in 10 mM Tris-HCl pH 7.5 and quantified by spectrophotometry. 1-2 ug of total RNA in 20 ul was mixed with an equal volume of 2X alkaline fragmentation solution (2 mM EDTA, 10 mM Na2CO3 and 90 mM NaHCO3) and incubated for 15 min at 95 C. The reaction was diluted by addition of 280 ul stop/precipitation solution (300 mM NaOAc pH 5.5, GlycoBlue Co-precipitant [Ambion, 15 mg/ml]), and fragmented RNA recovered by ethanol precipitation. Fragmented total RNA or RPFs (1-2 ug) were separated on 15% (wt/vol) denaturing polyacrylamide gels and RNA species migrating between 28 and 34 nt harvested. RNA was eluted from the gel slices on a rotator overnight at 4 C, in 600 ul RNA gel extraction buffer (300 mM NaOAc pH 5.5, 1 mM EDTA and 0.25% SDS). Eluted RNA was ethanol precipitated. The RNA samples from above were heated at 80 C for 2 min, cooled and the 3' phosphate group removed using T4 polynucleotide kinase (T4 PNK, New England Biolabs) at 37 C for 2 hrs in a 20 ul reaction lacking ATP. The RNA was concentrated by ethanol precipitation, resuspended in 10 mM Tris-HCl pH 7.5 and ligated in a 20 ul reaction overnight at 14 C to a preadenylated 3'-adaptor (5' rATGGAATTCTCGGGTGCCAAGG 3') using T4 RNA Ligase 2 truncated K227Q (New England Biolabs). This 3' adaptor was adenylated using a 5' DNA adenylation kit (New England Biolabs) following the manufacturers instructions. RNA was precipitated, loaded into a 15% (wt/vol) denaturing polyacrylamide gel and ligated RNA fragments migrating between 49 nt and 53 nt excised. The RNA was eluted, precipitated, resuspended as above and 5' phosphorylated using T4 PNK in the presence of 1mM ATP for 2 hrs at 37 C. RNA was concentrated by ethanol precipitation, resuspended in 10mM Tris-HCl pH 7.5 and ligated to a 5' RNA adaptor (5' rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrC 3') in a 20 ul reaction overnight at 14 C using T4 RNA Ligase (Promega). The fully adapted RNAs were recovered by ethanol precipitation, dissolved in 6 ul 10mM Tris-HCl pH 7.5 and 3 ul of this ligated product annealed to an RT primer (5' GCCTTGGCACCCGAGAATTCCA, 50 pmol) at 65 C for 5 min. The RNA was subsequently reverse transcribed at 55 C for 50 min in a 20 ul reaction following addition of first strand buffer (Invitrogen, to 1X), 2.5 mM MgCl2, 10 mM DTT, 0.5 ul SUPERase-In and 1 ul SuperScript III (Invitrogen), followed by heat inactivation at 85C for 5 min. For PCR and ribosomal depletion steps, see referenced manuscript.