Related to gene length - removing Intronic reads changes enrichment to splicing.

Discussion

Materials and Methods

Tissue preparation

Tissue samples were obtained from adult (postnatal day (P) 53-59)) male and female transgenic mice carrying a Cre transgene and a Cre-reporter transgene. Mice were anesthetized with 5% isoflurane and intracardially perfused with either 25 or 50 ml of ice cold, oxygenated artificial cerebral spinal fluid (ACSF) at a flow rate of 9 ml per minute until the liver appeared clear, or the full volume of perfusate had been flushed through the vasculature. The ACSF solution consisted of 0.5mM CaCl2, 25mM D-Glucose, 98mM HCl, 20mM HEPES, 10mM MgSO4, 1.25mM NaH2PO4, 3mM Myo-inositol, 12mM N-acetylcysteine, 96mM N-methyl-D-glucamine, 2.5mM KCl, 25mM NaHCO3, 5mM sodium L-Ascorbate, 3mM sodium pyruvate, 0.01mM Taurine, and 2mM Thiourea. The brain was then rapidly dissected and mounted for coronal slice preparation on the chuck of a Compresstome VF-300 vibrating microtome (Precisionary Instruments). Using a custom designed photodocumentation configuration (Mako G125B PoE camera with custom integrated software), a blockface image was acquired before each section was sliced at 250 μm intervals. The slice was then hemisected along the midline, and both hemispheres were then transferred to chilled, oxygenated ACSF. 
Each slice-hemisphere was transferred into a Sylgard-coated dissection dish containing 3 ml of chilled, oxygenated ACSF. Brightfield and fluorescent images between 4X and 20X were obtained of the intact tissue with a Nikon Digital Sight DS-Fi1 or a Sentech STC-SC500POE camera mounted to a Nikon SMZ1500 dissecting microscope. To guide anatomical targeting for dissection, boundaries were identified by trained anatomists, comparing the blockface image and the slice image to a matched plane of the Allen Reference Atlas. In general, three to five slices were sufficient to capture the targeted region of interest, allowing for expression analysis along the anterior/posterior axis. The region of interest was then dissected and both brightfield and fluorescent images of the dissections were acquired for secondary verification. The dissected regions were transferred in ACSF to a microcentrifuge tube, and stored on ice. This process was repeated for all slices containing the target region of interest, with each region of interest deposited into a new microcentrifuge tube. 
For whole cell dissociation, after all regions of interest were dissected, the ACSF was removed and 1 ml of a 2 mg/ml pronase in ACSF solution was added. Tissue was digested at room temperature (approximately 22°C) for a duration that consisted of adding 15 minutes to the age of the mouse (in days; i.e., P53 specimen had a digestion time of 68 minutes). After digestion, the pronase solution was removed and replaced by 1 ml of ACSF supplemented with 1% Fetal Bovine Serum (FBS). The tissue was washed two more times with the same solution and the sample was then triturated using fire-polished glass pipettes of decreasing bore sizes (600, 300, and 150 μm). The cell suspension was incubated on ice in preparation for fluorescence-activated cell sorting (FACS). FACS preparation involved adding 4’-6-diamidino-2-phenylindole (DAPI) at a final concentration of 4 μg/ml to label dead (DAPI+) versus live (DAPI-) cells. The suspension was then filtered through a fine-mesh cell strainer to remove cell aggregates. Cells were sorted by excluding DAPI positive events and debris, and gating to include red fluorescent events (tdTomato-positive cells). Single cells were collected into strip tubes containing 11.5μl of collection buffer (SMART-Seq v4 lysis buffer 0.83x, Clontech #634894), RNase Inhibitor (0.17U/µl), and ERCCs (External RNA Controls Consortium, MIX1 at a final dilution of 1x10-8) \cite{16179916,25150836}. After sorting, strip tubes containing single cells were centrifuged briefly and then stored at -80°C.
For nuclei isolation, dissected regions of interest were transferred to microcentrifuge tubes, snap frozen in a slurry of dry ice and ethanol, and stored at -80°C until the time of use.  To isolate nuclei, frozen tissues were placed into a homogenization buffer that consisted of 10mM Tris pH 8.0, 250mM sucrose, 25mM KCl, 5mM MgCl2, 0.1% Triton-X 100, 0.5% RNasin Plus RNase inhibitor (Promega), 1X protease inhibitor (Promega), and 0.1mM DTT.  Tissues were placed into a 1ml dounce homogenizer (Wheaton) and homogenized using 10 strokes of the loose dounce pestle followed by 10 strokes of the tight pestle to liberate nuclei . Homogenate was strained through a 30µm cell strainer (Miltenyi Biotech) and centrifuged at 900xg for 10 minutes to pellet nuclei. Nuclei were then resuspended in staining buffer containing 1X PBS supplemented with 0.8% nuclease-free BSA and 0.5% RNasin Plus RNase inhibitor. Mouse anti-NeuN antibody (EMD Millipore, MAB377, Clone A60) was added to the nuclei at a final dilution of 1:1000 and nuclei suspensions were incubated at 4°C for 30 minutes. Nuclei suspensions were then centrifuged at 400xg for 5 minutes and resuspended in clean staining buffer (1X PBS, 0.8% BSA, 0.5% RNasin Plus). Secondary antibody (goat anti-mouse IgG (H+L), Alexa Fluor 594 conjugated, ThermoFisher Scientific) was applied to nuclei suspensions at a dilution of 1:5000 for 30 minutes at 4°C. After incubation in secondary antibody, nuclei suspensions were centrifuged at 400xg for 5 minutes and resuspended in clean staining buffer. Prior to FACS, DAPI was applied to nuclei suspensions at a final concentration of 0.1µg/ml and nuclei suspensions were filtered through a 35µm nylon mesh to remove aggregates. Single nuclei were captured by gating on DAPI-positive events, excluding debris and doublets, and then gating on Alexa Fluor 594 (NeuN) signal. Strip tubes containing FACS isolated single nuclei were then briefly centrifuged and frozen at -80°C.