GSM3608775: Distinct RNAs associated with FH-LARP7; Homo sapiens; miRNA-Seq
The cells were lysed on ice for 10 min in buffer containing 50 mM HEPES pH 7.5, 150 mM NaCl, 2.5 mM MgCl2, 1% NP-40, protease inhibitors and RNase inhibitors. The lysate was passed 6 times through a 26G needle followed by water bath sonication. After sonication, the lysates were centrifuged for 20 min at 14,000 rpm and 4°C. The pre-equilibrated ANTI-FLAG M2 affinity gel (Sigma-Aldrich) was incubated with the lysates for 3 h on a head-over-tail rotor at 4°C. After incubation, beads were collected and were washed three times with washing buffer containing 50 mM HEPES pH 7.5, 300 mM NaCl and 2.5 mM MgCl2, followed by the last wash using 1 x PBS. FH-LARP7 and the interacting RNAs were eluted using 200 µg/mL 3x FLAG peptide (Sigma-Aldrich) in PBS. RNAs co-precipitated with FH-tagged LARP7 by anti-FLAG immunoprecipitations were purified with TRIzol reagent and separated on a denaturing polyacrylamide gel. RNAs were visualized under UV-light with the SYBR gold nucleic acid gel stain (Invitrogen) and three distinct bands were excised. The RNA was eluted overnight at 4°C in 300 mM NaCl and 2 mM EDTA and was subsequently precipitated. The RNAs were treated with the tobacco acid pyrophosphatase (Epicentre) to remove RNA cap structures. An adenylated adapter [5'-App-TGGAATTCTCGGGTGCCAAGG-(C7-amino)-3'] was ligated to the 3' end of the RNAs 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
GSM3608775