Am9342

Serial sections of the TMAs were cut into 5 µm FFPE sections and were mounted on SuperFrost Plus slides (Fisher Scientific, 12-550-15), air dried and baked overnight at 60 °C. Slides were then processed as specified by the NanoString GeoMx DSP Slide Preparation User Manual (NanoString Technologies, MAN-100 7). In brief, slides were dewaxed, underwent antigen retrieval and treated with proteinase K (Ambion, 2546) at 1 µg ml^–1 concentration. Slides were then post-fixed. For RNA probe hybridization, slides were placed in a slide rack with Kimwipes damped with 2× SSC lining the bottom. Each slide was treated with 200 µl of NanoString Technologies whole transcriptome RNA probe mix at a concentration of 4 nM per probe in 1× buffer R (NanoString Technologies). A Hybridslip (Grace Biolabs, 714022) was applied over each slide. Slides were incubated at 37 °C overnight. After hybridization, slides were dipped in a 2× SSC with 0.1% Tween 20 (Teknova, T0710) to remove the coverslips. They were then washed twice in 2× SSC and 50% formamide (ThermoFisher AM9342) at 37 °C for 25 min followed by two washes in 2× SSC for 5 min each at room temperature. Slides were blocked in buffer W (NanoString Technologies) at room temperature for 30 min, followed by the application of 200 µl morphology marker mix for 1 h. Details of the morphology markers are provided in Supplementary Table 7.

Neurons were plated on PDL-coated 28-mm glass bottom dishes (Invitrosci) and fixed 7 days later with 4% paraformaldehyde for 20 minutes at room temperature. Cells were permeabilized with cold 70% ethanol overnight at 4°C and washed the following morning for 5 minutes with wash buffer (25% formamide, 2X SSC). Neurons were stained with 40 20-nucleotide-long fluorescently labeled (Alexa-647) oligonucleotides designed against the Aag transcript overnight at 37°C in a heavily humidified chamber in hybridization buffer (100 mg/mL dextran sulfate, Sigma, D8906: 0.5 mg/mL E.coli tRNA, Sigma, R4251: 0.5 mg/mL ssDNA, Sigma D9156: 1 mg/mL Ultapure BSA, Ambion, AM2616: 10 mM VRC, New England Biolabs, S1402S: 25% formamide, Ambion, AM9342: 2X SSC, Ambion, AM9763). Finally, cells were stained with DAPI (2 μg/mL) in wash buffer for 30 minutes at 37°C before imaging. All the previous steps were done in nuclease-free solutions to avoid RNA degradation. We occasionally see faint transcripts in Aag^-/- cells due to initiation of endogenous transcription before hitting the inserted cassette that disrupts the gene.
Images were taken with a Hamamatsu ORCA-R² CCD Camera on a Zeiss Axio Observer.Z1 Microscope. 21 Z-stack images were taken, each 0.3 μm away from the previous. Images were compiled and foci per cell were counted with a MATLAB algorithm adapted from previously published methods [23 (link)].