GSM1608326: DT_total_2_2; Mus musculus; RNA-Seq
Liver tissue (~100 mg) was immersed in 2 mL of hypotonic buffer A [10 mM HEPES (pH 7.9), 2 mM MgCl2, and 2 mM KCl] supplemented with protease and phosphatase inhibitors (Thermo, 78441) and 40 U/mL ribonuclease inhibitor (Invitrogen, 10777-019). Thereafter, samples were ground with a loose (10 times) and tight (30 times) Dounce pestle and centrifuged at 2000 rpm for 5 min at 4oC. The supernatant (cytoplasmic fraction) was transferred to a fresh tube and the pellet (nuclei) was suspended in 0.5 mL of Buffer C [25% glycerol, 20 mM HEPES (pH 7.9), 2 mM MgCl2, 420 mM NaCl, and 0.2 mM EDTA] supplemented with protease, phosphatase, and ribonuclease inhibitors, incubated on ice for 15 min with mixing every 3 min, and centrifuged at 12000 rpm for 10 min at 4oC. The supernatant was diluted with three volumes of buffer A containing inhibitors (nuclear fraction) and kept on ice until IP was performed. Ten micrograms of an anti-hnRNPK antibody (#54) or control IgG (Vector Labs, I-1000) was conjugated to 50 µL of Dynabeads Protein-A (Life Technologies, 10002D) and used in a single IP reaction. Cytoplasmic and nuclear extracts were first incubated with IgG-conjugated Dynabeads for 30 min at 4oC with rotation, separated from the beads using a magnetic stand, and subjected to IP reaction with anti-hnRNPK antibody-conjugated Dynabeads under the same conditions. Beads were washed three times with IP buffer [150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, NP-40 (0.5% vol/vol), and Triton X-100 (1.0% vol/vol)] and RNA was eluted from the beads with 200 µL of RLT buffer supplemented with β-mercaptoethanol from an RNeasy Mini Kit (Qiagen). Thereafter, on-column DNase I digestion and RNA clean-up were performed according to the RNeasy Mini Kit protocol. The RNA concentration was measured using a Nanodrop instrument and RNA was stored at -80oC. RNA-sequencing library was prepared from 400 ng of total or immunoprecipitated RNA using an Illumina TruSeq RNA Sample Preparation Kit
GEO Accession
GSM1608326