Sample 7
Mus musculus
Protocols: L929 cells in 60-mm dishes were infected at an MOI of 10 in an initial volume of 1.5 ml DMEM with 1% FBS. After 1 h adsorption at 37 °C an additional 3.5 ml was added and incubation continued. At the appropriate time points, cells were treated with cycloheximide (Sigma-Aldrich; to 100 μg/ml; 2 min). Cells were rinsed with 5 ml of ice-cold PBS, the dishes submerged in a reservoir of liquid nitrogen for 10 s, transferred to dry ice and 400 μl of lysis buffer [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 μg/ml cycloheximide and 25 U/ml TURBO DNase (Life Technologies)] dripped onto the cells. 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. Cell lysates were subjected to Ribo-Seq and RNA-Seq. The methodologies employed were based on the original protocols of Ingolia and colleagues (Ingolia et al., 2009; Ingolia et al., 2012), except library amplicons were constructed using a small RNA cloning strategy (Guo et al., 2010) adapted to Illumina smallRNA v2 to allow multiplexing, as described previously (Chung et al., 2015).
ENA first public
2017-06-02
ENA last update
2016-11-03
External Id
SAMEA4534063
INSDC center alias
Department of Pathology, University of Cambridge
INSDC center name
Department of Pathology, University of Cambridge
INSDC first public
2017-06-02T17:02:10Z
INSDC last update
2016-11-03T11:12:21Z
INSDC status
public
Submitter Id
E-MTAB-5206:Sample 7
broker name
ArrayExpress
cell line
L929
cell type
fibroblast
common name
house mouse
growth condition
cycloheximide
protocol
Ribo-Seq
sample name
E-MTAB-5206:Sample 7