Journal: bioRxiv

Article Title: High-resolution scRNA-seq reveals genomic determinants of antigen expression hierarchy in African Trypanosomes

doi: 10.1101/2024.03.22.586247

Figure Lengend Snippet: a, Strategy to investigate VSG switching and template selection at the single cell level. b, Western blot analysis of Cas9 and γ-H2A protein expression in cells capable of doxycycline-inducible expression of Cas9 and a sgRNA targeting nucleotide site 152 of VSG-2 (sgRNA VSG-2.152). A wild-type cell line (Lister 427) and a cell line not transfected with the VSG-2 sgRNA but capable of inducible Cas9 expression (2T1 T Cas ) served as controls. EF1α served as a loading control. c, IFA of γ-H2A expression in cells transfected with sgRNA VSG-2.152 either before (Cas9(-)) or 4 hours after Cas9 induction (Cas9 (+)). γ-H2A was detected using an anti-γ-H2A antibody. Scale bar=10µm. d, FACS analysis of VSG-2 expression in sgRNA VSG-2.152 transfected cells in a time course until 96 hours post-Cas9 induction. VSG-2 expression was monitored using a fluorophore-conjugated (Alexa Fluor 488) anti-VSG-2 antibody. At least 10,000 events were analysed per sample. On the left panel, VSG-2 positive and VSG-2 negative cell lines are shown as controls.

Article Snippet: After washing the membranes 3 more times with PBS-T, the following HRP conjugated secondary antibodies were used: for Cas9 - anti-mouse (1:10,000 in 1% milk PBS-T, GE Healthcare, code NA931V); for γ-H2A - anti-rabbit (1:2000 in 1% milk/PBS-T, GE Healthcare, code NA934V); for EF1-α - anti-mouse (1:10,000 in 1% milk/PBS-T, GE Healthcare, code NA931V).

Techniques: Selection, Western Blot, Expressing, Transfection

Journal: bioRxiv

Article Title: High-resolution scRNA-seq reveals genomic determinants of antigen expression hierarchy in African Trypanosomes

doi: 10.1101/2024.03.22.586247

Figure Lengend Snippet: a-c, Growth curves following Cas9-based DSB induction for cut sites at nucleotide position 152 (a) and 782 (b) of VSG-2 CDS, and for cut sites at nucleotide position 609 and 1105 of VSG-8 CDS (c). The values shown represent the mean of three biological replicates for VSG-2.152 and VSG-2.782 cell lines. For VSG-8 cell lines one replicate was tested. e, Heatmap summarizing transcriptional signal end position on BES1 for recombinant switcher cells after a DSB in nucleotide position 782 of VSG-2 CDS (VSG-2.782) and nucleotide position 54824 of BES1 (BES1.54824). Each box in the heatmap represents the fraction of cells, for switchers to a given VSG (rows), which transcriptional signal ends at the given 5kb bin of BES1 (columns). All VSGs expressed in at least 10 cells were considered. d-h, Western blotting showing Cas9 and γ-H2a expression levels before and 24 hours post-Cas9 induction with doxycycline. Wild-type Lister 427 at 24 hours after doxycycline addition were used as a negative control. EF1α was used as a loading control. Clones used for scRNA-seq experiments are highlighted in bold. d, The results are shown for cut positions 152 and 782 in VSG- 2 CDS and two independent clones for cut position 609 in VSG-8 CDS. e, Cas9 and γ-H2a expression profiles for cut position 40225 (clones 1 and 2) in BES1 of a VSG-2 expressing cell line. f, Cas9 and γ-H2a expression levels for three clones of VSG-11 expressing cell line with cut position at 519. For Sanger sequencing analysis VSG-11.519 clone 1 was used. g, The blot shows the results for VSG-2 expressing cell lines with cut position at 54824 (clone 2) and 58149 (clones 1 and 2). h, The data is shown for VSG-11 expressing cell line with cut position at 729 (clones 1, 2, and 3) and VSG-2 expressing cell line with cut position at 54824 (clone 1). For Sanger sequencing VSG-11.729 clone 1 was used. Nucleotide positions are considered relative to the promoter in BES1 for cut sites

Article Snippet: After washing the membranes 3 more times with PBS-T, the following HRP conjugated secondary antibodies were used: for Cas9 - anti-mouse (1:10,000 in 1% milk PBS-T, GE Healthcare, code NA931V); for γ-H2A - anti-rabbit (1:2000 in 1% milk/PBS-T, GE Healthcare, code NA934V); for EF1-α - anti-mouse (1:10,000 in 1% milk/PBS-T, GE Healthcare, code NA931V).

Techniques: Recombinant, Western Blot, Expressing, Negative Control, Clone Assay, Sequencing