RNA-seq library construction: ~5000 NeuN+ nuclei were used to produce each RNA-seq library and two technical replicates were generated for each brain sample. ChIP was carried out using multiplexed MOWChIP-seq with MNase digestion for chromatin fragmentation and anti-H3K27ac (39135, Active Motif) antibody, following a published protocol (Zhu et al., Nature Protocols 14 (2019) 3366-3394.). One input per sample was generated using ~4,000 nuclei. The supernatant was discarded and the pellet was resuspended in 60 Вµl of PBS with freshly added 0.6 Вµl of PIC and 0.6 Вµl of PMSF and stored on ice until use for ChIP-seq.Д¬hIP-seq library construction: ~10,000 NeuN+ nuclei were used to produce each ChIP-seq library and two technical replicates were generated for each brain sample. Then, the sample was centrifuged at 1800 RCF at 4 вЊ for 15 min. The solution was mixed well and incubated on ice for 15 min. 200 Вµl of 1.8 M sucrose solution, 10 Вµl of 1 M CaCl2, and 3 Вµl of 1 M Mg(Ac)2 were added into the mixture. 200 Вµl of sorted NeuN+ nuclei suspension, containing ~26,000 nuclei, was added into 800 Вµl of ice-cold PBS for ChIP-seq experiment. The sorted NeuN+ nuclei were directly used for RNA-seq experiment. After incubation, the sample was sorted into NeuN+ and NeuN- fractions using a BD FACSAriaTM cell sorter (BD Biosciences).

The suspension was mixed well and incubated at 4 вЊ for 1 h on a rotator mixer (Labnet). To separate NeuN+ and NeuN- fractions, 6 Вµl of 2 ng/Вµl anti-NeuN antibody conjugated with Alexa 488 (MAB377X, Millipore) in DPBS was added into the nuclei suspension. Then, the supernatant was removed and 300 Вµl of 2%(w/v) normal goat serum (50062Z, Life technologies) in DulbeccoвЂ™s PBS (DPBS, Life technologies) was added to resuspend the nuclei pellet. The mixture was centrifuged at 10,000 RCF for 20 min. 500 Вµl of the sample was mixed with 750 Вµl of 50%(w/v) iodixanol (made by mixing 4 ml of OptiPrepTM gradient (Sigma-Aldrich) and 0.8 ml of diluent ).

The supernatant was discarded, and the pellet was resuspended in 0.5 ml of ice-cold NEB with freshly added 5 Вµl of PIC, 0.5 Вµl of PMSF, 0.5 Вµl of DTT, and 0.75 Вµl of RNase inhibitor. The cell suspension was centrifuged at 1000 RCF for 10 min. The homogenate was filtered with a 40 Вµm cell strainer (22-363-547, Thermo Fisher Scientific) and collected in a 15-ml centrifuge tube. The tissue was homogenized in tissue grinder (D9063, Sigma-Aldrich). One piece of mouse frontal cortex tissue (6-10 mg) was placed in 3 ml of ice-cold nuclei extraction buffer (NEB) with freshly added 30 Вµl of protease inhibitor cocktail (PIC, Sigma-Aldrich), 3 Вµl of 100 mM phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich) in Isopropyl alcohol, 3 Вµl of 1 M dithiothreitol (DTT, Sigma-Aldrich), and 4.5 Вµl of ribonuclease (RNase) inhibitor (2313A,Takara Bio). Nuclei extraction: All steps were conducted on ice, and all centrifugation was conducted at 4 вЊ. Neuron from the frontal lobes of the mouse cortexДЄntibody: anti-H3K27ac (39135, Active Motif) We are in the process of expanind our demo projects so you have more ideas and understand the many options of our msmac3D simulation and 3D Graphics programs.GEO help: Mouse over screen elements for information. New import/edit/display options for Ground surfaces:

Improvements on detection/separation flags to ignore 'proximity' without accelerations.

NOCOLL option to ignore interaction between Veh1/2 (so you can simulate variations of steer, etc on a single vehicle recontruction) has been extended to optionally allow/disallow ignoring veh2/3 and/or veh1/2 interaction.

Steering Ratio so you can input steering wheel angles instead of only front wheel steer angles.

larger underlay image sizes now allowed.

Improvements in the import and surface cloud type e57 files.

A surface visibility dialog is now available to easily turn on/off visibility (see below).

You can have many grounds of different types and reorder them, show/hide, turn on/off, clone, etc.

Clone ability for animations and Grounds.

Now have drag/drop positioning/repositioning for animated, animulated and simulated vehicles.

improvements to animulation option to allow easier editing.

More extensive animation capabilities (swap vehicle, change colors, etc, slider bar and sequential display, turn on/off, clone).2D animated and simulated vehicles can now follow the terrain graphically.Chart time editing.jpg (14.19 KiB) Viewed 1196 times

Veh2 samples