Mineral oil

For all viral injections, 2–3 month old mice were anesthetized with isofluorane (2%–5%) and placed in a stereotaxic apparatus (Kopf). Burr holes or craniotomies were made over targeted brain regions to permit pipette insertion. The pipettes (Drummond Scientific) were formed using a P-2000 laser puller (Sutter Instrument) to have a long tapered, 10–20 μm diameter tips. Pipettes were back-filled with mineral oil (Fisher Scientific) and then loaded with virus using a Nanoject II (Drummond Scientific). For identification of oxytocinergic inputs to the hippocampus, pAAV5-EF1α-DIO-ChETA-eYFP (1–1.2 μl; Penn Vector) was injected (0.1 μl/min) unilaterally into the PVN (from bregma in mm: 0.7 posterior, 0.25 lateral, 4 ventral) of Oxt-IRES-cre mice (from D. Olson and B. Lowell). For optogenetic manipulation of CA2 pyramidal cells, pAAV-EF1α-dflox.hChR2(H134R)-mCherry-WPRE-hGH (350 nl; Penn Vector) was injected (0.2 μl/min) bilaterally into CA2 (from bregma in mm: 1.6 posterior, ±1.6 medial/lateral, 1.7 ventral) of Amigo2-cre animals. After injection, the pipette remained in place for 5 min and was then slowly retracted. The scalp was then sutured shut, saline was administered subcutaneously, and buprenorphine (0.1 mg/kg) was administered subcutaneously for analgesia. Animals were allowed at least two weeks to recover to allow adequate expression of the virus.

Human plasminogen-free fibrinogen was purchased from Aniara Diagnostica (West Chester, OH, USA). Ovalbumin (98% purity, grade V) was purchased from Millipore-Sigma (St. Louis, MO, USA). Re-lipidated tissue factor (TF) (Dade Innovin, B4212–40 Siemens Healthcare, Erlangen, Germany), acetic acid (glacial, 100%, reagent grade), mineral oil (light, C₁₆H₁₀N₂Na₂O₇S₂, reagent grade), and 96-well microplates (polystyrene, flat bottom, non-tissue culture treated plate, Falcon) were purchased from Fisher Scientific (Hampton, NH, USA). Human alpha thrombin was purchased from Haematologic Technologies (Essex Junction, VT, USA). Recombinant tissue-type plasminogen activator (tPA) was from Innovative Research (Novi, MI, USA). Distillated water (diH₂O) was prepared using a Direct Q3 system (Millipore-Sigma, St. Louis, MO, USA). Tris Buffered Saline (10x, 500 mM Tris, 1500 nM NaCl) was prepared in house and the pH was adjusted to 7.4. Synthetic phospholipid vesicles (PS:PE:PC ratios of 15:41:44) were a gift from Dr. Dougald Monroe, University of North Carolina. For kinetic optical density measurements, a Synergy H1 spectrophotometer (BioTek, Winooski, VT, USA) was used.

LSD1/CoREST/nucleosomes complexes were reconstituted by mixing hLSD1(171–852, R608A,N717A,D721A)/hCoREST(286–440) protein complex with Xenopus nucleosomes containing Widom 601 nucleosome positioning DNA at a molar ratio of 3.3:1 enzyme:nucleosome in 10 mM Tris-Cl pH 7.5, 75 mM NaCl, 1 mM DTT. The histone H3K4M mutant was used to enhance binding of LSD1 to the nucleosome. The LSD1/CoREST/nucleosome was purified by Superdex 200 size exclusion chromatography in the reconstitution buffer supplemented with 0.1 mM PMSF. Pooled fractions were concentrated to ~10 mg/ml in VS500 centrifuge ultrafiltration devices (Vivascience).
Purified complexes were crystallized at 4°C using the modified microbatch procedure (D’Arcy et al., 2004 (link)) with 1 μl of ~10 mg/ml LSD1/CoREST/nucleosome complex mixed with 1 μl of 25 mM HEPES pH 7.5, 75 mM ammonium citrate, 10% PEG2000-MME (Fluka) and overlaid with 70 μl of Al’s oil [1:1 mixture of silicon oil (Clearco) and mineral oil (Fisher)]. The crystals were soaked in 25 mM Bis-Tris pH 6.0, 75 mM ammonium citrate, 6% PEG2000-MME before a 50% PEG smear mixture of 10% PEG400, 10% PEG350-MME, 10% PEG550-MME, 10% PEG750-MME and 10% PEG500-DME was added in 4% increments at room temperature every 5 minutes to a final concentration of 24% (Chaikuad et al., 2015 (link)).