Meso-seq for in-depth transcriptomics in ultra-low amounts of FACS-purified neuronal nuclei

Daily light-induced transcription in visual cortex neurons drives downward Firing Rate Homeostasis and stabilizes sensory processing

“…Grip2 ) ( Figure 1B,C and Supplemental Data Table 1 ). Consistent with previous findings on sensory-induced gene expression in the mouse visual cortex 37,39,57 and further indicating the validity of our transcriptomic data, our analyses also revealed that the RNA levels of many housekeeping genes do not change substantially across a single day (e.g. ActB , Htt , Ubc ; Supplemental Figure 1E ).…”

“…95.3% and 85.8% of all excitatory neurons respectively express Fos and Npas4 at ZT1). When we conducted similar RNAscope smFISH experiments for Igf1, a gene that we did not identify in our transcriptomic analyses as regulated but that we and others have previously identified as a late-induced effector gene in a sparse subtype of GABAergic interneurons that express the vasoactive intestinal peptide (VIP INs 28,37,39,57 ), we found that also this gene is induced every day in a light-dependent manner (Supplemental Figure 1H). Thus, our bulk transcriptomic analyses likely underestimate the total number of daily (light-)regulated genes in the visual cortex since they lack the cell-type-specificity to detect all daily (light-induced) changes in gene expression in the various subtypes of visual cortex neurons.…”

2P-NucTag: on-demand phototagging for molecular analysis of functionally identified cortical neurons

“…The sequence of the cloned plasmid was validated by Sanger sequencing and GCaMP7f + 4xAla, P2A and H2B-PAmCherry were all found to be in frame. The plasmid was packaged into AAVDJ at a viral titer of 7.26E+15 essentially as described 46 .…”

“…Our approach combines large-scale in vivo two-photon (2P) functional imaging of cortical PNs with reliable and selective 2P phototagging of nuclei in a subset of neurons based on their functional properties. Using fluorescence-activated cell sorting (FACS) to isolate phototagged neuronal nuclei post hoc , combined with our recently developed Meso-seq approach for transcriptomics in ultra-sparse populations 46 , we achieve previously unattainable molecular characterization of functionally identified PNs in vivo in behaving animals.…”

“…To determine whether place cells and silent cells differed in their gene expression programs, RNA-seq was performed on both populations of sorted nuclei by Meso-seq, an approach that enables reliable identification of differentially expressed genes in ultra-low amounts for FACS-isolated neuronal nuclei (i.e., tens of sorted nuclei, Fig. 3D) 46 . After FACS-isolating the photo-tagged nuclei, libraries were generated with the Meso-seq protocol and were sequenced at a depth of 40-60 million reads per library (fig.…”