Mus musculus
Protocols: Ribosome Immunoprecipitation (IP): Samples were extracted from mice, flash-frozen in liquid nitrogen and stored at −80 °C until use. 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.7 antibody (H9658, Sigma) or 10µg of mouse monoclonal IgG1 antibody (Merck, 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 a cold room at 4 °C. After up to 12 h, samples were washed three times with high-salt buffer (50 mM Tris, pH 7.4, 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 and RNA was extracted with Dynabeads mRNA Direct. mRNA was captured with Dynabeads oligo(dT) (Life Technologies) according to the manufacturer's guidelines. We used a derivation of MARS-seq 58. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a 2200 TapeStation instrument.
ENA-FIRST-PUBLIC
2023-01-20
ENA-LAST-UPDATE
2023-01-20
External Id
SAMEA112302650
INSDC center alias
University College London UK Dementia Research Institute
INSDC center name
University College London UK Dementia Research Institute
INSDC first public
2023-01-20T00:18:10Z
INSDC last update
2023-01-20T00:18:10Z
INSDC status
public
Submitter Id
E-MTAB-12515:Sample 6
age
12 to 16
broker name
ArrayExpress
cell_type
perivascular macrophage
common name
house mouse
organism part
cerebral hemisphere
sample name
E-MTAB-12515:Sample 6
sex
male
strain
Lyve1ncre:Cx3cr1ccre