Mouse glutamatergic neurons immunofluorescence images upon Suz12 isoforms deletions
Description
The Polycomb repressive complex 2 (PRC2) mediates epigenetic maintenance of gene silencing in eukaryotes via methylation of histone H3 at lysine 27 (H3K27). Alternative splicing (AS) of the PRC2 core component SUZ12 generates an uncharacterised isoform SUZ12-S, which co-exists with the canonical SUZ12-L isoform in the same cell in virtually all tissues and developmental stages. While SUZ12-S is necessary and sufficient to ensure correct repression of Polycomb target genes via promoter-proximal H3K27me3 deposition, SUZ12-L is needed for maintaining global H3K27 methylation levels. We showed that mouse embryonic stem cells (ESCs) lacking either of the two isoforms have a slower exit from pluripotency and fail to acquire neuronal cell identity upon differentiation stimuli. This dataset contains raw immunofluorescence images of ESCs derived neurons stained for H3K27me3 (RRID:AB_2614987), MAP2 (RRID:AB_2138153), and TUBB3 (RRID:AB_2564645). All images were counter-stained with DAPI before mounting.
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Mouse ESCs differentiation to glutamatergic neurons was performed as previously described in Bibel et al. (2007) Nat. Protoc. Briefly, on day zero the 4 million ESCs were seeded in low attachment dishes cultured in DMEM high glucose media supplemented with 10% FBS, 1% Non-essential amino acids, 1% GlutaMAX, 1% Sodium-pyruvate, and 1% penicillin/streptomycin to allow the formation of embryoid bodies. On day 4 5μM retinoic acid was added to the culture media. On day 8 embryoid bodies were either collected for RNA-seq or dissociated by enzymatic digestion with TrypLE and seeded on poly-D-lysine and laminin coated glass coverslips and cultured in DMEM high glucose media supplemented 1% N2 and 2% B27 without vitamin A supplements. On day 13 neuronal cells were collected for immunofluorescence. Coverslips were fixed in 3% formaldehyde aqueous solution for 20 minutes at room temperature. Formaldehyde was subsequently quenched with 30mM glycine solution for 5 minutes and coverslips were washed 3 times with PBS. Cells were permeabilised with ice-cold methanol for 5 minutes and washed 3 times with 0.05% Tween-20 in PBS. Cells were blocked for 1 hour with 2% goat serum in PBS. Primary antibodies were incubated overnight in a humidified chamber at 4ºC with the following dilutions: H3K27me3 (Active Motif, 61017) 1:300; TUBB3 (Biolegend, 802001) 1:1000; MAP2 (abcam, 5392) 1:2500. Cells were washed 3 times and then incubated for 1 hour at room temperature with the following alex-fluorophore coupled secondary antibodies diluted. Cells were counterstained with DAPI for 5 minutes and mounted on glass cover slides. Images were acquired using a confocal microscope (LLSM980 Airy Scan 2, Zeiss) with 40X magnification. Images of 2 tiles were acquired with a z-stack of 6 slices of 0.5µm in a range of 2.8µm. Nuclear areas were quantified in Fiji using maximal z-stack projection of both DAPI and H3K27me3 and regions of interest (nuclei) were selected using a combination of manual selection and Weka Segmentation tool. When importing the images in Fiji following the instructions as reported in the 2 attached screenshots: image 1 is TUBB3; image 2 is MAP2; image 3 is H3K27me3; image 4 is DAPI.