title: SST-GFP Nuclei (5000), Rep 1
design: A dorsal segment from the L3-L5 region from each SSTGFP or Sun1-GFP(fl/+) mouse was dissected using small spring scissors and snap frozen on dry ice for later processing. On the day of experiment, the tissue segment was placed in 1 mL of Nuclear Extraction Buffer (NEB) supplemented with RNase and Protease Inhibitors (20 mM Tris HCl pH 8, 5 mM MgCl2, 25 mM KCl, 250 mM Sucrose, 40 U/mL RNasin Plus (Promega), 1 tablet/10 mL Protease Cocktail Mini EDTA-Free (Roche), 1 uM DTT (Sigma), 0.3% NP-40 (Pierce)). Dounce Homogenization (10 strokes Pestle A, 10 strokes Pestle B) was performed to liberate the nuclei using a 2 ml homogenizer (Sigma). The homogenate was filtered through a 50 m Partec filter (Sysmex) into a regular (not low-binding) 1.7 ml microcentrifuge tube (Axygen), since low-binding tubes create loose pellets that are easily displaced.  The filter was washed with an additional 700 l NEB and the homogenate was then centrifuged for 10 mins at 4C (500g) to form a loose pellet. The pellet was resuspended with 500 ul of NEB using a p1000 pipettor with regular-bore tips and 10 aspiration/dispense cycles, and filtered through a 20 um Partec filter into a 5 ml polypropylene tube. DAPI (4',6-diamidino-2-phenylindole) was added to the sample to a final concentration of 5 ng/ml. We made several modifications to the INTACT procedure by Mo et al. (Mo et al., 2015) to simplify the workflow and make the procedure suitable for small spinal cord samples: (1) All volumes and vessels were scaled down to accommodate the smaller size of a dorsal lumbar spinal segment from mouse compared to cortex; (2) Density gradient separation (e.g. Iodixanol) was removed, as we noticed it caused clumping of nuclei and added no additional benefit; (3) FANS, as opposed to bead immunoprecipitation, was used in  order to most specifically and cleanly isolate the relatively low number of GFP+ nuclei in the dorsal segment. FANS was performed using a BD FACSAria II sorter (BD Biosciences) using a pressure of 35 pounds per square inch (psi) and a 100 um sort nozzle. Gates were established to capture singlet nuclei with high DAPI staining intensity. For SSTGFP, 5000 DAPI+/GFP+ events were sorted into 350 ul of RNAaqeous Micro (Life Technologies) lysis buffer. For the total nuclei control sample from Sun1-GFP(fl/+) mice, 5000 DAPI+/GFP- events were isolated in the same manner. FANS data were analyzed using FlowJo 3.0 (FlowJo LLC). For each mouse line, n=3 samples were isolated. Captured samples were placed on ice and immediately processed for RNA isolation. RNA isolation was performed using the RNaqeuous Micro kit (Life Technologies) according to the manufacturers instructions. DNase digestion was not performed at this step, since the downstream library preparation included a DNase step. For library preparation, all samples were processed at the same time using the SoLo RNA-Seq kit (NuGen Technologies) according to the manufacturers instructions in a PCR-clean laminar flow hood. For the PCR amplification step, 17 cycles were used, as this was the optimal number of cycles determined in a prior qPCR optimization assay according to the manfacturers instructions. Library concentration was assessed with the Qubit 2.0 fluorometer and dsDNA HS assay (Life Technologies), checked for quality on the Bioanalyzer (Agilent) and then run on the HiSeq 2500 (Illumina) using a 50 base-pair, single-end read protocol.