Extended Data Fig. 1. Optimization of BARseq2 for detecting endogenous mRNAs.

(A) Relative sensitivity (means and individual data points) of BARseq2 in detecting Slc17a7 using the indicated fixation times, normalized to that achieved with 5 mins of fixation. n = 3 for 480 mins and n = 4 for other conditions. (B) Rolony counts for Slc17a7 using either random primers or specific primers at two different concentrations. The two concentrations used were 5 μM (low) and 50 μM (high) for random primers, and 0.5 μM (low) and 5 μM (high) for specific primers. Lines indicate means and dots/crosses represent individual samples. n = 2 slices for each condition. (C) (D) BARseq2 sensitivity compared to RNAscope. (C) Spot density detected by BARseq2 or RNAscope in each 100 μm bin along the laminar axis in auditory cortex. Error bars indicate standard errors. The dashed line indicates linear fit for Slc30a3 and Cdh13. Slope = 1.65 and R² = 0.73. n = 5 slices for both BARseq2 and RNAscope. (D) shows the means and individual samples for each gene. (E)(F) Positions of rolonies across five sequencing cycles using the original (E) or the optimized (F) sequencing protocol. Scale bars = 10 μm. (G) The distribution of minimum distance between rolonies imaged in the first cycle and in the fifth cycle using the original or the optimized protocol. (H) Median distance between rolonies imaged in the indicated cycles and the closest rolonies imaged in the first cycle using the original or the optimized protocol. Error bars indicate standard errors. For both (G) and (H), n = 148,708 rolonies for optimized condition and n = 12,114 for original condition. (I)(J) The distribution of absolute rolony intensities for the first sequencing cycle (I) and relative rolony intensities after 6 sequencing cycles and one stripping step, normalized to the intensities in the first sequencing cycle (J). Amino-allyl dUTP concentrations used are indicated. In (I), n = 63,852 rolonies for 0.08 μM and n = 4,286 rolonies for 0.5 μM; in (J), n = 128,976 rolonies for 0.08 μM and n = 113,235 rolonies for 0.5 μM.