Journal: bioRxiv

Article Title: Cell-type specific in vitro gene expression profiling of stem-cell derived neural models

doi: 10.1101/2020.04.30.064709

Figure Lengend Snippet: GFP and mCherry protein accumulation in HEK293 cells transduced with PTRE-RiboTag lentivirus were confirmed by (A) microscopy and (B) Western blot. Lysate – total soluble protein. Sup – supernatant. IP – immunoprecipitated protein

Article Snippet: Primary antibodies include alkaline phosphatase conjugated anti-Flag (Sigma-Aldrich A9469) and anti-HA antibodies (Immunoreagents #MuxOt-111-DALP), biotin conjugated anti-HA (Biolegend 901505) and anti-V5 (Invitrogen MA5-15253-BTIN) antibodies, and unconjugated anti-GFP (Origene TA150070), anti-mCherry (Novus NBP2-25158).

Techniques: Transduction, Microscopy, Western Blot, Immunoprecipitation

Journal: bioRxiv

Article Title: Cell-type specific in vitro gene expression profiling of stem-cell derived neural models

doi: 10.1101/2020.04.30.064709

Figure Lengend Snippet: (A) Total soluble protein from co-culture lysates, supernatants (Sup), and immunoprecipitated (IP) protein were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to HA or V5. (B) mCherry and GFP mRNA concentration was measured in triplicate by reverse transcription digital PCR in RNA isolated from the co-culture lysates (yellow), HA IP (blue), and V5 IP (purple), and plotted as a ratio. (C) RPL22-V5 and RPL22-HA were simultaneously IPed from co-culture lysates and selectively eluted in the indicated order using V5 or HA peptides, respectively. Total soluble protein from co-culture lysate and IPed protein were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to V5 or HA. (D) RPL22-V5 and RPL22-HA were sequentially IPed in the indicated order from co-culture lysates. Total soluble protein from co-culture lysates and IPed protein were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies to HA or V5.

Article Snippet: Primary antibodies include alkaline phosphatase conjugated anti-Flag (Sigma-Aldrich A9469) and anti-HA antibodies (Immunoreagents #MuxOt-111-DALP), biotin conjugated anti-HA (Biolegend 901505) and anti-V5 (Invitrogen MA5-15253-BTIN) antibodies, and unconjugated anti-GFP (Origene TA150070), anti-mCherry (Novus NBP2-25158).

Techniques: Co-Culture Assay, Immunoprecipitation, SDS Page, Concentration Assay, Digital PCR, Isolation

Journal: bioRxiv

Article Title: Cell-type specific in vitro gene expression profiling of stem-cell derived neural models

doi: 10.1101/2020.04.30.064709

Figure Lengend Snippet: (A) mCherry and GFP mRNA concentration was measured in triplicate by reverse transcription digital PCR in RNA isolated from co-culture lysates (yellow), HA IP (blue), and V5 IP (purple), and plotted as a ratio. (B) RNA-seq reads were mapped to a hybrid reference genome containing hg38 and mm20 chromosomes and quantified by species for monocultures, co-culture, and IP samples. (C) Spearman correlation plot of RNA-seq for co-culture and IP samples. (D) The change in uniquely mapping reads (IP – input) for each chromosome was plotted against the initial (Input) percent uniquely mapping reads. Each dot represents a chromosome (hg38 – blue, mm20 – purple). First and second IPs are represented by open and filled dots, respectively. Chromosomes that fall above the red dotted line are enriched whereas chromosomes that fall below the red dotted line were depleted in IP samples compared to the Input. (E) The change in on-target (e.g. hg38 for HA IP and mm20 for V5) percent uniquely mapping reads (IP – Input) was plotted against the initial (Input) percent uniquely mapping reads at the gene level. Protein coding genes are in cyan and non-protein coding genes in pink. First and second IPs are represented by circles and triangles, respectively. Mitochondrially encoded genes have additional black colored fill.

Article Snippet: Primary antibodies include alkaline phosphatase conjugated anti-Flag (Sigma-Aldrich A9469) and anti-HA antibodies (Immunoreagents #MuxOt-111-DALP), biotin conjugated anti-HA (Biolegend 901505) and anti-V5 (Invitrogen MA5-15253-BTIN) antibodies, and unconjugated anti-GFP (Origene TA150070), anti-mCherry (Novus NBP2-25158).

Techniques: Concentration Assay, Digital PCR, Isolation, Co-Culture Assay, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Cell-type specific in vitro gene expression profiling of stem-cell derived neural models

doi: 10.1101/2020.04.30.064709

Figure Lengend Snippet: The ratio of mCherry to GFP transcript abundance was measured in triplicate from pre- and post-IP samples and reported as a ratio of the average concentration. IPs were performed in both orders as indicated.

Article Snippet: Primary antibodies include alkaline phosphatase conjugated anti-Flag (Sigma-Aldrich A9469) and anti-HA antibodies (Immunoreagents #MuxOt-111-DALP), biotin conjugated anti-HA (Biolegend 901505) and anti-V5 (Invitrogen MA5-15253-BTIN) antibodies, and unconjugated anti-GFP (Origene TA150070), anti-mCherry (Novus NBP2-25158).

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Cell-type specific in vitro gene expression profiling of stem-cell derived neural models

doi: 10.1101/2020.04.30.064709

Figure Lengend Snippet: (A-B) RPKM of reporter gene expression (GFP/mCherry) in primary mouse astrocytes and hiPSC-derived motor neurons split by (A) RiboTag and (B) cell type. RPKM was calculated using reads mapped to GFP or mCherry and a total library size of reads mapping to the on-target cell type. (C) RNA-seq reads were mapped to a hybrid reference genome containing hg38 and mm20 chromosomes and quantified by species for co-cultures and IP samples. Matched samples are indicated by replicate number. (D) mCherry and GFP levels were measured by RNA-seq. The ratio of mCherry to GFP is reported for co-cultures and IP samples from hiPSC-MNs (MN IP) and primary mouse astrocytes (mAstro IP). HA RiboTag (purple). V5 RiboTag (blue). Lines indicate matched IPs and co-cultures.

Article Snippet: Primary antibodies include alkaline phosphatase conjugated anti-Flag (Sigma-Aldrich A9469) and anti-HA antibodies (Immunoreagents #MuxOt-111-DALP), biotin conjugated anti-HA (Biolegend 901505) and anti-V5 (Invitrogen MA5-15253-BTIN) antibodies, and unconjugated anti-GFP (Origene TA150070), anti-mCherry (Novus NBP2-25158).

Techniques: Expressing, Derivative Assay, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Cell-type specific in vitro gene expression profiling of stem-cell derived neural models

doi: 10.1101/2020.04.30.064709

Figure Lengend Snippet: (A) Scatter plot of on-target reporter (RPL22-HA-GFP; RPL22-V5-mCherry) gene expression in RPKM versus depletion efficiency. RPKM was calculated using the library size for the indicated cell type (i.e. only human reads were used to calculate RPKM for reporter expression in hiPSC-MNs and vice versa). (B) Scatter plot of the initial species composition of the coculture (left – human; right – mouse) versus the corresponding depletion efficiency for every IP. The on-target cell type is indicated by color (mAstro – purple; hiPSC-MN – blue) and shape indicates the RiboTag (HA – circles, V5 – triangles). For example, blue circles & triangles on the left examine the relationship between the depletion efficiency and the relative composition of the on-target cell type (human) in the initial coculture. The purple circles on the left examine the relationship between the depletion efficiency and the off-target cell type (mouse). The reverse is true for the plot on the right.

Article Snippet: Primary antibodies include alkaline phosphatase conjugated anti-Flag (Sigma-Aldrich A9469) and anti-HA antibodies (Immunoreagents #MuxOt-111-DALP), biotin conjugated anti-HA (Biolegend 901505) and anti-V5 (Invitrogen MA5-15253-BTIN) antibodies, and unconjugated anti-GFP (Origene TA150070), anti-mCherry (Novus NBP2-25158).

Techniques: Expressing

Journal: bioRxiv

Article Title: Cell-type specific in vitro gene expression profiling of stem-cell derived neural models

doi: 10.1101/2020.04.30.064709

Figure Lengend Snippet: Plotted is the ratio of mCherry to GFP across co-cultures (yellow), V5 IPs (blue), and HA IPs (purple) for (A) NGN2-induced excitatory neuron (iGluN) and ASCL1/DLX2-induced GABAergic neuron (iGaNs) co-cultures prepared from a single differentiation and RiboTag transduction per cell type, and (B) primary mouse astrocytes (mAstro) and hiPSC-MN co-cultures across independent replicates. Lines indicated matched IPs and co-cultures.

Article Snippet: Primary antibodies include alkaline phosphatase conjugated anti-Flag (Sigma-Aldrich A9469) and anti-HA antibodies (Immunoreagents #MuxOt-111-DALP), biotin conjugated anti-HA (Biolegend 901505) and anti-V5 (Invitrogen MA5-15253-BTIN) antibodies, and unconjugated anti-GFP (Origene TA150070), anti-mCherry (Novus NBP2-25158).

Techniques: Transduction