title: GSM4193023: Sall1-CreER:Ribotag_MG_Sort_7; Mus musculus; RIP-Seq
library_construction_protocol: microglia isolation: Mice were anesthetized with Pental (1:2 in PBS) and were perfused with PBS. Brains were dissected, crudely chopped and incubated for 20 min at 37°C in a 1 ml HBSS solution containing 2% BSA, 1 mg/ml Collagenase D (Sigma) and 50µg/ml DNase1 (Sigma). In the middle of incubation homogenates were pipetted for further dissociation. Next, the homogenates were filtered through a 150μm mesh, washed with cold FACS buffer (2% FCS, 1mM EDTA in PBS-/-) and centrifuged at 2200 RPM, at 4°C, for 5 min. For the enrichment of microglia, the cell pellet was re-suspended with 3ml of 40% percoll solution and centrifuged at 2200 RPM, no acceleration and breaks, at RT for 15 min. Next, the cell pellet was resuspended, passed through 80µm mesh, washed with 5ml FACS buffer and centrifuged at 1400 RPM at 4°C for 5 min, followed by antibody (Ab) labeling and flow cytometry analysis. immunoprecipitation: Mice were anesthetized with Pental (1:2 in PBS) and were perfused with PBS. Samples were homogenized on ice in ice-cold homogenization buffer (50 mM Tris, pH 7.4, 100 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM DTT, 1:100 protease inhibitor (Sigma), 200 units/ml RNasin (Promega) and 0.1 mg/ml cycloheximide (Sigma) in RNase free DDW) 10% w/v with a Dounce homogenizer (Sigma) until the suspension was homogeneous. To remove cell debris, 1 ml of the homogenate was transferred to an Eppendorf tube and was centrifuged at 10,000g and 4°C for 10 min. Supernatants were transferred to a fresh Eppendorf tube on ice, then 10μl was removed for 'input' analysis and 10µg of anti-HA antibody (H9658, Sigma) or 10µg of mouse monoclonal IgG1 antibody (Sigma, Cat# PP100) was added to the supernatant, *followed by 4 h of incubation with slow rotation in a cold room at 4 °C. Meanwhile, Dynabeads Protein G (Thermo Fisher Scientific), 100 μl per sample, were equilibrated to homogenization buffer by washing three times. At the end of 4 h of incubation with antibody, beads were added to each sample, followed by incubation overnight in cold room at 4 °C. After not more than 12 h, samples were washed three times with high-salt buffer (50 mM Tris, 300 mM KCl, 12 mM MgCl2, 1% NP-40, 1 mM DTT, 1:200 protease inhibitor, 100 units/ml RNasin and 0.1 mg/ml cycloheximide in RNase free DDW), 5 min per wash in a cold room on a rotator. At the end of the washes, beads were magnetized and excess buffer was removed, 100µl Lysis Buffer  was added to the beads (Ambion Life Technologies). For Sort+aHA IP samples (RiboTag IP after sorting), 30,000 microglial cells were sorted directly into 100µl Lysis Buffer (Ambion Life Technologies) containing 10µg of anti-HA antibody (H9658, Sigma) or 10µg of mouse monoclonal IgG1 antibody (Sigma, Cat# PP100), the rest of IP was continued as above from *. PolyA-tailed mRNA was captured using the Dynabeads mRNA DIRECT kit (Ambion Life Technologies) according to manufacturer's guidelines. RNA-seq libraries were constructed used a derivation of MARS-seq as described in Jaitin et al. (2014) adapted for bulk cell populations.
attributes.tag: GEO Accession
attributes.value: GSM4193023