Because some of the EST libraries were derived from unspecified tissues or under artificially modified expression conditions, we removed 1,898 such human libraries (out of 8,145; 23.3%) and 211 such mouse libraries (out of 841; 25.1%) from our analysis (see Methods) and organized the rest into a hierarchy of manually curated tissue/organ classes.
Of the >14,500 unique tags identified in both libraries, 224 had a modified expression level; they are known to participate in inflammation, ion transport, signal transduction, oxidative stress, apoptosis, metabolism, and catabolism.
Thus, we generated two independent miRNA expression libraries ("control library" and "HD library").
Genomic fragments containing human miRNA precursors were cloned into a modified pLL3.7 vector, and construction of the human miRNA expression library has been described previously (Zhou et al., 2013).
Here, we report on the construction and application of a random small RNA expression library for use in identifying small interfering RNA (siRNA) effectors that can modify complex cellular phenotypes in mammalian cells.
Here, we report on the development and application of a convergent random small RNA expression library for use in mammalian cell types to isolate novel RNAs capable of modifying complex cellular phenotypes.
HeLa or COS7 cells Culture medium: DMEM (Dulbecco's Modified Eagle Medium) (Gibco, cat. No 11965-092) supplemented with 10% Fetal Calf Serum, 1% MEM Nonessential Amino Acid solution (Gibco), 20mM HEPES (pH 7.2), 1% Penicillin- streptomycin (Gibco cat. No 15140-122), 1mM sodium pyruvate (Sigma) Phosphate Buffered Saline (PBS) FuGene6 transfection reagent (Roche) cDNA expression library.
The second core peptide sequence was modified to create libraries.
We found numerous cases where a given nucleotide position was likely modified in both libraries.
There remains the possibility of some artificially modified libraries escaping from this screening, but their effect on the present analysis should be minimized, not to mention that some of them may in fact equalize the expression count, thus making detection of differential expression more stringent.
