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. 2021 Dec 17;10:e66973. doi: 10.7554/eLife.66973

Figure 1. Chromatin landscape in differentiating Purkinje neurons.

(A) Schematic of Purkinje cell (PC) differentiation and growth at P0, P7, and adult (approximately 8-week-old) timepoints. EGL – external granule layer, PCL – Purkinje cell layer, IGL – internal granule layer, MCL – molecular cell layer, GCL – granule cell layer. (B) Immunofluorescence staining with Calb1 (green) of PCs in murine cerebella at P0, P7, and adult timepoints. (C) Example of PC nuclei stained with Itpr1 (green) post-dissociation and pre-sorting, counterstained with DAPI. (D) Representative plots of fluorescence-activated nuclear sorting of PCs at P0, P7, and adult timepoints with Itpr1. (E) Workflow schematic of nuclei isolation, antibody staining with anti-Itpr1, fluorescence-activated sorting, and downstream sequencing applications. (D) Integrated genome viewer (IGV) representation of example regions of differentially regulated genes (Olfr cluster – always silent, Cacnb4 – always expressed, Grid1 and Pde1c – developmentally down-regulated, Atp2a3 and Cep76 – developmentally up-regulated). Top tracks show RNA expression in RPKM (reads per kilobase per million mapped reads), mCG tracks show methylation level in CG context from 0 to 0.8, hmCG tracks show hydroxymethylation level in CG context from 0 to 0.45, mCH and hmCH show methylation and hydroxymethylation level in CH context (H = A, C, or T) from 0 to 0.04. ATAC tracks show ATACSeq read density in RPKM from 0 to 10. H3K4me3 tracks show input normalized enrichment in RPKM from 0 to 5. H3K27me3 tracks show input normalized enrichment in RPKM from 0 to 3.

Figure 1.

Figure 1—figure supplement 1. Quality control of sequencing datasets.

Figure 1—figure supplement 1.

(A) Immunofluorescence staining of Purkinje cells at P0, P7, and adult timepoints with Calb1 (green) and Itpr1 (red). (B) Integrated genome viewer (IGV) representation of Purkinje-specific markers (Itpr1, Calb1, Car8, Kcnip1, Kcnma1, Pcp2) enrichment and depletion of granule (Gabra6, Etv1, Cbln1, Neurod1, Atp2b1, Kcnd2) and glial (Gfap, Sorcs3, Aldh1l1, Olig2) markers. (C) Pearson correlation of RNASeq datasets. (D) Pearson correlation of gene body accumulation of 5hmCG + 5 mCG (bisulfite sequencing [BSSeq]), 5hmCG, and 5mCG (derived from maximum likelihood estimator model of BSSeq and oxidative bisulfite sequencing [OxBSSeq]). (E) Pearson correlation of assay for transposase-accessible chromatin sequencing (ATACSeq), H3K4me3, and H3K27me3 chromatin immunoprecipitation sequencing (ChIPSeq) dataset enrichment ±5 kb around the gene promoters. (F) Pearson correlation of gene body accumulation of 5hmCH and 5mCH (derived from maximum likelihood estimator model of BSSeq and OxBSeq). (G) Bisulfite conversion and oxidation efficiency in BSSeq and OxBSSeq datasets. (H) IGV representation of BSSeq (showing 5hmCG + 5 mCG signal together) and OxBSSeq (showing 5hmCG + 5 mCG signal separately) at the three developmental timepoints.