Saturation curves for each library.

T. thermophila (Tet1; A, B), Heterocapsa sp. (Het; C, D) and P. tricornutum (Pha, E, F) illustrating how many unique transcripts (A, C, E) and genes (B, D, F) that were detected as a function of sequencing depth. The sequencing depth (number of read pairs) are plotted on the x-axes and the unique transcripts or unique gene counts on the y-axes.</p

Cells of <i>Heterocapsa</i> sp. and <i>P</i>. <i>tricornutum</i> imaged after different ways of fixation.

Cells of Heterocapsa sp. and P. tricornutum imaged after different ways of fixation.</p

Bioanalyzer traces of final libraries showing product length when cells were fixed with formaldehyde and methanol.

Upper and lower columns correspond to the same cells, collected at the same time points. (PDF)</p

Top 10 most highly expressed genes (<i>i</i>.<i>e</i>. reference transcripts) in single-cell libraries of <i>Heterocapsa</i> sp. (Het) and <i>T</i>. <i>thermophila</i> (Tet1 and Tet2).

Top 10 most highly expressed genes (i.e. reference transcripts) in single-cell libraries of Heterocapsa sp. (Het) and T. thermophila (Tet1 and Tet2).</p

Reagents and combinations used to test lysis on optimization slides.

Reagents and combinations used to test lysis on optimization slides.</p

Number of expressed genes and unique transcripts per spot for different spot categories.

Background (empty spots), singles (one cell), doubles (two cells) and clusters (>2 cells) are identified based on the microscopic images and MASC-seq sequencing libraries of A) T. thermophila (two libraries; Tet1 and Tet2), B) Heterocapsa sp. (Het) and C) P. tricornutum (Pha). An example image of a spot is shown above each spot category and species. The number of spots (n) for each category and library are given. Significance levels of Wilcoxon rank-sum tests comparing number of unique transcripts per spot between pairs of spot categories are indicated, with * = p-value < 0.05; ** = p-value < 0.0001; and NS = non-significant.</p

Scatter plots illustrating correlations between sequencing libraries.

Each circle represents one gene, and its position indicates its total counts (number of unique transcripts) in the two libraries compared. A-C) T. thermophila. Bulk RNA added to the array vs. MASC-seq data (cells) for Tet1 (A) and Tet2 (B). MASC-seq data for Tet1 vs Tet2 (C). D) Bulk RNA added to the array vs. MASC-seq data for Heterocapsa sp. E) Bulk RNA added to the array vs. MASC-seq data for P. tricornutum. Pearson correlation coefficients (r) are given for each subplot.</p

Overview of the MASC-seq method.

A) Samples are collected as described in the Methods section and fixed. Eukaryotic plankton that have hard cell wall covering undergo freeze-thaw cycling in liquid nitrogen to pre-permeabilize cell walls. Subsequent steps are attachment to the slide, staining, imaging and permeabilization. After permeabilization, polyadenylated transcripts leaking out of the cells are captured by the surface probes, which function as primers in the following reverse transcription reaction. Cells are removed, cDNA is enzymatically cleaved off, harvested, amplified and processed for Illumina sequencing (path to the right in panel A). C) The slides have six separate arrays that can accommodate different experiments. Each array contains ~2000 spots with oligo-dT probes that have spot-specific barcodes (IDs) and UMIs. D) During cDNA synthesis, a cell positioned on top of a certain spot will get the spot-specific ID incorporated into its cDNA that can later be linked to the image of the spot. For optimization purposes, a special type of optimization arrays with oligo-dT probes covering the whole surface (B) can be used. Here, the reverse transcription is conducted using fluorescently labeled nucleotides, and after cell removal, the slide is scanned and the fluorescence cDNA footprint will correspond to the amount of cDNA synthesized (path to the left in panel A). Library preparation and sequencing are not conducted in this case. E) Optimizations conducted to make the MASC-seq protocol more suitable for single-celled eukaryotic plankton. From top to bottom: tested fixatives, formaldehyde (2%) and methanol (absolute) both gave positive results on optimization array slide, methanol produced longer final library molecules. Two different enzymes (pepsin and lysozyme) and acid (0.01 M HCL) were tested for cell permeabilization and only pepsin gave positive results. Freeze-thaw cycling in liquid nitrogen and room temperature (3 cycles) improved the amount of product in the final libraries. A cell attachment test was performed to estimate cell loss during the different steps of the protocol. Fig illustrations in A, B and D are redrawn with permission from [29], Springer Nature Limited.</p

S4 Fig -

Examples of A) T. thermophila cells prefixed with methanol, attached to the slide and stained with hematoxylin and eosin; B) T. thermophila cells prefixed with methanol and then freeze-thawed in liquid nitrogen and room temperature water (3 times, attached on the slide and stained with hematotoxylin and eosin). (PDF)</p

Bioanalyzer traces of final libraries before and after freeze-thaw cycling.

Upper and lower columns correspond to the same cells, collected at the same time points. (PDF)</p