GSM1356265: ribosome footprinting nmf205-/-; n-Tr20J/J rep1; Mus musculus; RNA-Seq
Tissue homogenization was performed with a mixer mill (Retsch MM400) in 1.0 ml of 20 mM Tris-Cl (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% (v/v) Triton X-100, 25 U/ml Turbo DNase I, 100 mg/ml cycloheximide, and 2 mg/ml harringtonine. RNase I-treated lysates were ultracentrifuged through a sucrose cushion to obtain the ribosome pellet from which RNA was extracted. 26 nt to 34-nt RNA fragments were purified by electrophoresis on 15% polyacrylamide TBE-urea gel. Linker addition and cDNA generation and circularization were then performed. Subtraction of rRNA with a pool of oligos was performed as indicated in the protocol. The resulting DNAs were amplified with different indexing primers to distinguish each sample as indicated in the protocol. The libraries were analyzed by sequencing on an Illumina HiSeq 2000 platform.
GEO Accession
GSM1356265