Extended Data Fig. 4: RiboTag.
From: eIF2α controls memory consolidation via excitatory and somatostatin neurons
a, Schematic representation of the AAV-EF1α-DIO-Rpl22-3HA-IRES-YFP-WPRE-hGH polyA (AAV-DIO-RiboTag) viral vector. We developed a Cre-dependent RiboTag viral vector based on AAV-DIO-RiboTag41. In the presence of Cre recombinase under a Camk2a promoter, the Rpl22-HA-IRES-YFP cassette is rearranged to the ON configuration that allows the Rpl22-HA expression under the Eef1a promoter in CAMK2α-positive excitatory neurons. b, Representative illustration of target area for bilateral AAV-DIO-RiboTag injection in the dorsal hippocampus. Tagged ribosomes from Rpl22-HA-expressing CAMK2α-positive excitatory neurons were recovered from total lysate by immunoprecipitation (IP) with magnetic beads conjugated to anti-HA antibodies. RNAs were extracted from the IP and total lysate. c, Immunohistochemistry for HA staining demonstrating that expression of AAV-DIO-RiboTag viral vector is restricted to the CAMK2α-positive excitatory neurons. Two independent experiments showed similar results. Scale bars: 200 μm. d–g, Comparison of RNA-Seq from IP to input showed an enrichment for excitatory neurons marker Camk2a and depletion of oligodendrocytes marker 2′, 3′-cyclic-nucleotide 3′ phosphodiesterase (CNPase, Cnp), astrocytes marker (Gfap) and markers of inhibitory neurons Sst and Pvalb, indicating a successful immunoprecipitation from excitatory neurons.
