Illumina HiSeq 2000 sequencing; Examples of ribosome profiling in chlamydomonas and mouse
Examples of ribosome profiling in chlamydomonas and mouse
Murine 17 clone 1 cells were maintained in Dulbeccos modification of Eagles medium supplemented with 10% (vol/vol) fetal calf serum. Cells (107) were plated in a 10 cm dish and upon reaching 100% confluence, cycloheximide (Sigma-Aldrich) was added to 100 ug/ml. After 2 min, cells were rinsed with 5 ml of ice-cold PBS, the dishes submerged in a reservoir of liquid nitrogen for 10 sec, transferred to dry ice and 400 ul of lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 ug/ml cycloheximide and 25 U/ml TURBO DNase (Life Technologies) dripped on. The cells were scraped extensively to ensure lysis, collected and triturated with a 26-G needle ten times. Lysates were clarified by centrifugation for 20 min at 13,000 g at 4 C, the supernatants recovered and stored in liquid nitrogen. For mouse samples, lysates were slowly thawed on ice and 250 ul treated with 7.5 ul RNase 1 followed by incubation for 45 min at room temperature on a rotating wheel. 10 ul of SUPERase-In RNase inhibitor was added, the sample layered onto a 1M sucrose cushion in mammalian polysome buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 ug/ml cycloheximide) and ultracentrifuged at 28,000 rpm (16 hr, 4 C) in a Beckman SW55Ti rotor. Subsequently, all ribosome pellets were resuspended in 200 ul of the corresponding polysome buffer and digested with proteinase K (10 mM Tris-HCl pH 7.5, 10% SDS, 200 ug/ml Proteinase K [New England Biolabs]) for 30 min at 42 C. RPFs were recovered by extracting twice with pre-warmed (65 C) acidic phenol:chloroform (Life Technologies) and once with chloroform (1:1, vol/vol, buffered with 10 mM Tris pH 7.5, 0.1 mM EDTA), followed by ethanol precipitation. RPFs were resuspended in 10 mM Tris-HCl pH 7.5 and quantified by spectrophotometry. 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.