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| sample_name |
DRS002066 |
| sample comment |
2 à 107 THP-1 cells were lysed using mirVana Kit lysis buffer (600 µl). The cell lysate was diluted in 10à IP buffer (25 mM Tris-HCl pH7.5, 150 mM KCl, 1 mM DTT, 1à protease inhibitor, 0.1% NP40). The mixture was centrifuged 14,000 rpm for 15 min at 4 °C. The supernatant were divided into three tubes, two of them for the immunoprecipitation, while the third tube is for IP library negative control. 10 µg of mouse monoclonal antibody against 7mG and 2,2,7mG caps (American research product cat#03-57028, clone H20) (P Bochnig et al. 1987, European Journal of Biochemistry / FEBS, 168(2), pp.461â467), 10ug of mouse monoclonal antibody against 7mG and 2,2,7mG caps clone K121 (Calibochem cat#D00036157) (Krainer 1988, Nucleic Acids Research, 16(20), pp.9415â9429) and (1 ml of RNase-DNase free water (Gibco) for the negative control) were mixed with 1 ml of cell lysate in the presence of 40 units of RNaseOUT and rotated over night at 4 °C. 100 µl of washed Dynabeads protein G (cat#100.03 Invitrogen) were added and the tubes were rotated at 40 °C for an extra 5 h. G beads were washed 3 à 10 min at 4 °C with 200 µl IP buffer, resuspended in 200 µl IP buffer and then supplemented with 50 µg Protease K. RNA was separated from the beads by incubating at 40 °C for 30 min, extracted with phenol/chloroform and ethanol precipitated. The RNAs recovered for both IP and negative control were then dephosphorylated and decapped, and used to prepare libraries based on 5' adapter ligation (Kawano et al. 2010, BioTechniques, 49(4), pp.751â755). In addition, to capture sRNA that are not capped or sRNAs that have a 5' phosphate, control libraries were prepared from 1.6 µg of THP-1 small RNA extracted by the mirVana (Ambion). All RNAs except for the control library were dephosphorylated by Shrimp Alkaline phosphatase (SAP, USB) according to manufacture recommendation, and then dephosphorylated with 1 unit of TAP (Epicentre) in the presence of 20 units of RNaseOUT. The de-capped RNA was recovered by extraction with phenol/chloroform and ethanol precipitated. |
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