TRAP-seq of astrocytes in the APP NL-F knock-in model of ß-amyloidopathy
TRAP-seq of astrocytes in the APP NL-F knock-in model of ß-amyloidopathy
Tissue was lysed in ice-cold lysis buffer (in mM: 20 HEPES, 10 MgCl2, 150 KCl, 0.5 DTT) along with 100 μg/mL cycloheximide (Sigma), cOmplete ULTRA protease inhibitors (Sigma) and RNAse inhibitors (Superasin – Life Technologies; RNAsin – Promega). Cell lysate was centrifuged to clear debris and solubilised with 1% NP40 and 30 mM 1,2-diheptanoyl-sn- glycero-3-phosphocholine (DHPC – Avanti Polar Lipids).  Following further centrifugation, supernatants were added to pre-prepared anti-GFP (19C8 and 19F7 antibodies – Sloan Kettering Memorial Centre) coated magnetic beads (Dynabeads MyOne Streptavadin T1 – Life Technologies). Following immunoprecipitation overnight at 40C with continuous rotation, beads were washed four times. Cell-type specific mRNA, attached to immunoprecipitated GFP-tagged ribosomes, was isolated and purified using the Agilent Nanoprep kit. TRAP-sequencing was performed utilising the Clonetech - SMART-Seq v4 Ultra Low input RNA library preparation.