NextSeq 500 sequencing; Ribosome profiling of the retrovirus murine leukemia virus
Ribosome profiling of the retrovirus murine leukemia virus
In order to produce infectious virions, the full-length MuLV Moloney strain proviral clone pNCA (Colicelli J and Goff SP 1988, a kind gift of S.P. Goff, Columbia University) (GenBank accession number: NC_001501) was transfected to 293T monolayers at 40% confluence. Cells were harvested at 72 h post-transfection and tissue culture supernatant was used to infect in duplicate fresh Rat2 (Rattus norvegicus) monolayers at 10% confluence. At 96 h p.i. cells were treated with cyclohexamide [CHX] (Sigma-Aldrich; to 100 mg/ml; 2 min), or harringtonine HAR (LKT laboratories; 2 mg/ml, 3 min) then CHX (to 100 mg/ml; 2 min). Cells were rinsed with 5 ml of ice-cold PBS, the dishes were submerged in a reservoir of liquid nitrogen for 10 s and then transferred to dry ice and 400 ml of lysis buffer [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 mg/ml cycloheximide and 25 U/ml TURBO DNase (Life Technologies)] dripped onto the cells. Cells were scraped, collected and triturated with a 26-G needle ten times. Lysates were clarified by centrifugation for 20 min at 13,000 g at 4ºC. Cell lysates were subjected to RiboSeq and RNASeq as described in (Chung 2015). The methodologies employed were based on the original protocols of Ingolia and colleagues (Ingolia 2009 and Ingolia 2012), except ribosomal RNA contamination removed by treatment with duplex-specific nuclease (DSN) and library amplicons were constructed using a small RNA cloning strategy adapted to Illumina smallRNA v2 to allow multiplexing.
Experimental Factor: infect
mock
Experimental Factor: compound
none
Experimental Factor: protocol
RNA-seq