Syn per reagent

Synaptosomes were extracted using Syn-PER™ reagent (Thermo Scientific), following manufacturer protocol. Briefly, animals were anaesthetised with pentobarbital and then perfused with RNase free, ice-cold PBS. Amygdalae were dissected and weighed. Tissue samples were homogenised in 10 volumes of the Syn-PER™ reagent followed by centrifugation (1200 × g for 10 min at 4 °C). The pellets containing cell debris were discarded and synaptosomal fraction was concentrated by subsequent centrifugation (15,000 × g for 20 min at 4 °C). Resulting highly enriched synaptosomal fractions were used for RNA extractions.

Neurons were lysed in ice-cold 1× RIPA buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 1% Na-deoxycholate, 0.1% SDS, and 1% NP-40) and supplemented with 1:100 protease inhibitor cocktail (Sigma-Aldrich). Lysed neurons were sheared by sonication, cell debris pelleted at 15000 rpm for 5 min at 4°C, and the clarified supernatant transferred to a prechilled 1.7 ml microcentrifuge tube. Total cell extracts were denatured at 95°C for 5 min, using either home-made 5× Laemmli buffer, BOLT 4× Sample buffer (Life Technologies) and BOLT 10× reducing agent (Life Technologies), 2×-, or 4× Laemmli sample buffer (both from Bio-Rad). To obtain synaptic fractions, cell pellets were treated with 1 ml ice-cold Syn-PER reagent (Thermo Scientific catalogue number 87793) and supplemented with 1:100 protease inhibitor cocktail (Sigma-Aldrich). Neurons were incubated on ice for 10 min, gently triturated 10 times, centrifuged at 1200 × g for 10 min at 4°C, and supernatant was transferred to ice-cold microcentrifuge tubes, then recentrifuged at 15,000 × g for 20 min at 4°C. The pellet was then resuspended in 20 µl 2× Laemmli sample buffer (Bio-Rad). Acid-extracted histone fractions were prepared using a protocol described previously (Shechter et al., 2007 (link)). Histone pellets were denatured using 2× Laemmli sample buffer (Bio-Rad).

Animals were sacrificed and brain tissue dissected on ice as described above. Hippocampi and cortices from a single hemisphere were homogenized in 200 μl and 400 μl, respectively, Syn-PER Reagent (Thermo Scientific #87793) with protease and phosphatase inhibitors using a polypropylene pellet pestle. Homogenates were centrifuged at 1200×g for 10 min at 4 °C. Supernatants were collected and spun again at 15,000×g for 20 min at 4 °C. The crude synaptosomal (P2’) pellets were resolubilized in RIPA with 1% SDS and boiled in Laemmli sample buffer with 5% β-mercaptoethanol and 1% SDS at 95 °C for 5 min.

Synaptosomes were extracted using Syn-PER Reagent as per manufacturer instructions with some modifications (Thermo Scientific, USA)^{48 (link),49 (link),60 (link)}. Briefly, 50 mg of brain tissue was used from each sample for synaptosome extraction in 1 ml of Syn-PER Reagent. Tissues were homogenized slowly by Dounce glass homogenization on ice with ~10 slow strokes. The resulting tissue homogenates were transferred to a centrifuge tube. Samples were centrifuged at 1400×g for 10 minutes at 4 °C to remove the leftover tissue debris. After centrifugation, the supernatant was transferred to a new tube. Again, supernatant (homogenate) was centrifuged at high-speed 15,000 × g for 20 min at 4 °C. The supernatant was removed as a cytosolic fraction and synaptosomes recovered in the pellet form. Both the cytosolic fraction and synaptosome pellet were processed for RNA and protein extraction. The synaptosome pellet was also processed for transmission electron microscopic (TEM) analysis.

Synaptosomes were isolated using Syn-PER Reagent (ThermoFisher Scientific, Waltham, MA) according to the manufacturer’s protocol. Briefly, approximately 30 mg of tissue was homogenized using Dounce glass homogenizer in the presence of Halt Protease Inhibitor Cocktail (ThermoFisher Scientific, Waltham, MA) and Phosphatase Inhibitor Cocktail (MilliporeSigma, Burlington, MA). The homogenate was spun down at 1200×g for 10 min at 4 °C. The supernatant was centrifuged at 15,000×g for 20 min at 4 °C to obtain the pellet of synaptosomes. The pellet was then resuspended in HBK (HEPES-buffered Krebs-like) buffer as described before [24 (link)]. The concentration of synaptosomes was determined using flow cytometry. The samples were stored at − 80 °C until use. Synaptosome preparations are routinely analyzed by Western blot and electron microscopy to ensure the quality of the preparation, as we have previously reported [24 (link)].