GSM3608776: HEK293 Input; Homo sapiens; miRNA-Seq
Polyclonal anti-LARP7 antibodies from rabbit serum SY7862 were coupled overnight at 4°C to Protein A sepharose beads. Cells were washed with cold PBS and lysed in 1.5 ml of lysis buffer [25mM Tris (pH 7.5),150mM KCl, 0.5% (v/v) NP-40, 2mM EDTA, 1mM NaF, 1mM DTT and 0.5mM AEBSF]. The lysates were cleared by centrifugation at 17,000xg for 30 min, before addition to the beads. After 3 h of rotation at 4°C, the beads were washed four times with wash buffer [50mM Tris–HCl (pH 7.5), 300mM KCl, 1mM MgCl2 and 0.5% (v/v) NP-40] and once with PBS. For RNA extraction 200 µl Proteinase K buffer [200mM Tris (pH 7.5), 300mM NaCl, 25mM EDTA, 2% SDS] containing 0.2 mg/ml Proteinase K were added. The samples were incubated 30 min at 50°C, the RNA was then isolated with phenol/chloroform/isoamyl alcohol. Input samples were prepared by extracting total RNA directly from an aliquot of the lysates using TRIzol according to the manufacturer's instructions. In short, an adenylated adapter [5'-App-TGGAATTCTCGGGTGCCAAGG-(C7-amino)-3'] was ligated to the 3' end of the total RNA and RNA co-precipitated in anti-LARP7 immunoprecipitations by a truncated T4 RNA Ligase 2. Subsequently, the 5' RNA-adapter (5'-GUUCAGAGUUCUACAGUCCGACGAUC-3') was ligated using the T4 RNA Ligase 1 (NEB). After reverse-transcription using a specific primer, the cDNA was amplified by PCR, gel-fractionated and bands or regions above empty adapter dimers were cut out. PCR products from these gel slices were eluted in elution buffer, precipitated and dissolved in water. The quality of the libraries was assessed by Bioanalyzer measurements.
GEO Accession
GSM3608776