brainphys neuronal culture media sm1 kit  (STEMCELL Technologies Inc)

Journal: bioRxiv

Article Title: MAPT expression is mediated by long-range interactions with cis -regulatory elements

doi: 10.1101/2023.03.07.531520

Figure Lengend Snippet: a) Loop plot of HiCCUPs nominated loops to the MAPT promoter. Top panel: Nominated loops by dataset. Top: HiC loops to the MAPT promoter from iCell GlutaNeuron cultures. Middle: Capture-C loops to the MAPT promoter from iCell GlutaNeuron cultures. Bottom: Capture-C loops to the MAPT promoter from iCell GABANeuron cultures. Bottom panel: Top: Track of ENCODE annotated candidate CREs. Middle: ATAC-seq peaks from KOLF2.1J-hNGN2 differentiated neurons. Bottom: H3K27ac and CTCF ChIP-seq peaks from Day 14 BrainPhys differentiated neurons. b) Proportion of HiC global loop calls by ENCODE annotation. c) Proportion of Capture-C MAPT promoter loops by ENCODE annotation for both iCell GlutaNeurons and GABANeurons.

Article Snippet: On day 3 neurites were present, and cells were renewed with Cortical Neuron Culture Media: BrainPhys neuronal medium (StemCell Technologies), 50X SM1 supplement (StemCell Technologies), 10 μ g/mL BDNF (StemCell Technologies), 10 μ g/mL NT-3 (StemCell Technologies), and 1 mg/mL Mouse Laminin (Gibco).

Techniques: Capture-C, ChIP-sequencing

Journal: bioRxiv

Article Title: MAPT expression is mediated by long-range interactions with cis -regulatory elements

doi: 10.1101/2023.03.07.531520

Figure Lengend Snippet: a) Heatmap showing expression of MAPT and cell type marker genes in NPCs, Day 14 BrainPhys differentiated neurons (BrainPhys Neurons), KOLF2.1J-hNGN2 Neurons (NGN2 Neuron), and iCell GlutaNeurons. b) Differential Capture-C directional p-value track. Top panel: Comparison of NPCs and Day 14 BrainPhys Neurons. Middle panel: Comparison of iCell GlutaNeurons (Excitatory) and iCell GABANeurons (Inhibitory). c) Zoom in of chr17: 45,225,000–45,320,000. Top panels: Chromatin accessibility across cell types generated by snATAC-seq in DLPFC nuclei. Bottom panels: Differential capture-C comparing BrainPhys differentiated neurons and NPCs with the neuron-specific region highlighted in gray.

Article Snippet: On day 3 neurites were present, and cells were renewed with Cortical Neuron Culture Media: BrainPhys neuronal medium (StemCell Technologies), 50X SM1 supplement (StemCell Technologies), 10 μ g/mL BDNF (StemCell Technologies), 10 μ g/mL NT-3 (StemCell Technologies), and 1 mg/mL Mouse Laminin (Gibco).

Techniques: Expressing, Marker, Capture-C, Comparison, Generated

Journal: bioRxiv

Article Title: MAPT expression is mediated by long-range interactions with cis -regulatory elements

doi: 10.1101/2023.03.07.531520

Figure Lengend Snippet: a . Boxplots showing statistically significant (*p<0.05, ANOVA with Fisher’s LSD) elements tested in luciferase assays in iCell GlutaNeurons. b. Boxplots showing statistically significant (*p<0.05, ANOVA with Fisher’s LSD) elements tested in luciferase assays in KOLF2.1J-hNGN2 neurons. c. RT-qPCR of MAPT expression. Barplots show statistically significant regions tested in Day 14 BrainPhys differentiated neurons by CRISPRi experiments. Red asterisk indicates regions significant by 3’ mRNA-seq and RT-qPCR. Gray asterisk indicates regions significant in either RT-qPCR or 3’ mRNA-seq. (*p<0.05, ANOVA with Fisher’s LSD). d. Heatmap showing differential expression of MAPT and all genes expressed (cpm > 30) within 1 Mb of MAPT TSS. Left y-axis indicates the distance of the midpoint of the target region tested in CRISPRi experiments from the MAPT promoter (chr17:45894000). e. Browser view of significant nominated regions. Green: iCell GlutaNeuron luciferase assay. Blue: KOLF2.1J-hNGN2 neuron luciferase assay. Teal: CRISPRi. Red: significant in luciferase and CRISPRi. f. – i. Key CREs regulating MAPT expression. Zoom in on regions validated by qPCR and CRISPRi ( f. −652,338, g. −464,677 & −461,949, h. −44,905, i. 48,416). Top panel: Normalized Accessibility from KOLF2.1J-hNGN2 differentiated Neurons. Bottom panel: Tracks of design region, luciferase region (Green: tested in iCell Gluta Neurons; Blue: tested in KOLF2.1J-hNGN2 neurons; *p<0.05, ANOVA with Fisher’s LSD), gRNA position, and nominated regions (DLPFC Links: Blue: control-specific link; gray: common link; Red: AD-specific link).

Article Snippet: On day 3 neurites were present, and cells were renewed with Cortical Neuron Culture Media: BrainPhys neuronal medium (StemCell Technologies), 50X SM1 supplement (StemCell Technologies), 10 μ g/mL BDNF (StemCell Technologies), 10 μ g/mL NT-3 (StemCell Technologies), and 1 mg/mL Mouse Laminin (Gibco).

Techniques: Luciferase, Quantitative RT-PCR, Expressing, Control