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Fig. 3 | Genome Biology

Fig. 3

From: ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics

Fig. 3

Cryopreservation and RNA integrity of ACME-dissociated cells. a Experimental workflow of ACME dissociation for cytometry and RNA analysis. be Flow cytometry profiles of singlet gated (FSC) Schmidtea mediterranea ACME-dissociated cells, stained with DRAQ5 (DNA) and Concanavalin-A (cytoplasm), directly after dissociation (b), after a first freezing step (c), after FACS (d), and after a second freezing step (e). DRAQ5 scales are shown in linear values to focus on G1 and G2 populations and differ due to the freezing steps. Aggregates are gated out by FSC. Percentages relative to the number of total singlets are shown in black for debris and cells. G1 (red) and G2 (blue) percentages refer to these population proportions. G1 and G2 proportions do not greatly vary, but FACS sorting effectively enriches these populations. f Flow cytometry profiles of singlet gated (FSC) S. mediterranea ACME-dissociated cells after 1 to 5 freezing cycles. DRAQ5 and Concanavalin-A-positive cells are shown in green and debris in black. Scale and gating conditions as in b and c. gk Bioanalyzer profiles and RIN values of RNA samples. Inferred values (Inf RIN, blue open boxes) are calculated from a correlative analysis of the % area of the two ribosomal bands compared to the total as shown in Additional file 1: Figure S2. As a control sample, we used RNA from worms directly in TRIzol (g). A size ladder is displayed, and the two major RNAs (18S and 28S) are indicated. Time- and temperature-dependent RNA degradation were tested keeping samples at room temperature (h) or on ice (i) for 6 h. RNA integrity along the protocol was tested for the conditions described in be (j), showing partial degradation after FACS. RNA integrity was tested after 1 to 5 freeze/thaw cycles (k)

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