Journal: Life Science Alliance

Article Title: High-quality single-cell transcriptomics from ovarian histological sections during folliculogenesis

doi: 10.26508/lsa.202301929

Figure Lengend Snippet: (A) Schematic representation of different cDNA amplification methods for single cells isolated from alcohol- and formalin-fixed ovarian sections. (B) PCA of single oocytes and granulosa cells isolated from mouse ovarian sections analyzed with DRaqL-SC3-seq. Representative histological images are shown to the right of the plots, and isolated cells are indicated with dashed lines. Note that granulosa cells that exhibited a mixed transcriptome profile with oocytes were excluded from subsequent analyses. (C) Real-time PCR analysis of cDNAs amplified from single granulosa cells isolated from alcohol-fixed ovarian sections. Ct values of Arbp in cDNAs amplified with the indicated methods are represented with boxplots. P -values by a t test are also shown. (D) Schematic representation of DRaqL combined with protease treatment. (E) Real-time PCR analysis of cDNAs amplified with different Smart-seq2-based cDNA amplification methods from single granulosa cells isolated from formalin-fixed ovarian sections. The Arbp C t values are represented with boxplots. P -values by a t test are shown above the graph. (F) Scatterplots of the averages and SDs of the Arbp C t values in cDNAs amplified with the indicated methods from alcohol- and formalin-fixed sections. The DRaqL-adapted methods are linked with their original methods with dashed lines. The average and SD for DRaqL-Protease-Smart-seq2 and Smart-seq2 from formalin-fixed sections were calculated for the three independent experiments shown in . (G) PCA of single oocytes and granulosa cells isolated from the formalin-fixed mouse ovarian sections. (B) The color coding is the same as in (B). Source data are available for this figure.

Article Snippet: First, we examined the use of DRaqL in conjunction with a SMART-Seq v4 3′DE Kit (Takara Bio), a commercially available Smart-seq2-based kit that allows multiplex cDNA library preparation by including index sequences in the reverse transcription primers.

Techniques: Amplification, Isolation, Real-time Polymerase Chain Reaction

Journal: Life Science Alliance

Article Title: High-quality single-cell transcriptomics from ovarian histological sections during folliculogenesis

doi: 10.26508/lsa.202301929

Figure Lengend Snippet: (A) Application of DRaqL to SMART-Seq v4 3′DE Kit (Takara Bio) with different SDc concentration in the cell lysis buffer. Boxplots of Ct values of indicated genes in amplified cDNAs from 10-pg mESCs are shown. 0.31% and 0.63% SDc in the cell lysis buffer were quenched by 7.6% Triton X-100 before cDNA amplification. (B) Adaptation of SMART-Seq v4 3′DE Kit with DRaqL evaluated with real-time PCR. Boxplots of Ct values of indicated genes in amplified cDNAs from 10-pg mESCs are shown. cDNAs were amplified with the SMART-Seq v4 3′DE Kit added with different combinations of reverse transcriptases (SuperScript II, SuperScript III, and Maxma H minus) upon application to DRaqL, with 0.31% SDc in cell lysis buffer (n = 6, 4, 8, 6, 6, and 4, respectively). The combination of SuperScript II and SuperScript III showed the best performance with DRaqL. (C) Boxplots of Ct values of Arbp in cDNAs amplified with indicated methods (DRaqL-SC3-seq, DRaqL-Smart-seq2, GEO-seq , and Smart-3SEQ for frozen section ) for single cells isolated from alcohol-fixed cell-block sections, evaluated with real-time PCR (n = 48, 12, 48, and 10, respectively). “Smart-3SEQ starting from fresh-frozen tissue on Arcturus LCM” and GEO-seq were performed as described ( ; ). The differences from the original protocols were as follows: Cells were isolated with Zeiss PALM MB4 in this study, instead of the Leica CellCut System and Arcturus laser-capture microdissection : Single cells were subjected to GEO-seq cDNA amplification in this study, instead of about 10–20 cells in the original protocol . (D) Boxplot of the number of detectable genes (estimated ≥1 copy expression) in granulosa cells in alcohol-fixed sections amplified with DRaqL-SMART-Seq v4, SMART-Seq v4, DRaqL-Smart-seq2, and Smart-seq2. P -values of a t test between DRaqL-adapted and original methods were also indicated. (E) Optimization of the time length for protease inactivation in the DRaqL-Protease-Smart-seq2 cDNA amplification for single granulosa cells from formalin-fixed sections. Boxplots show the Arbp Ct values. (F) Boxplots of the Arbp Ct values in the DRaqL-Protease-Smart-seq2 from single granulosa cells in formalin-fixed ovarian sections in three independent experiments (n = 12, 24, and 8). Dataset #1 is the dataset shown in . The results of cDNA amplifications with the Smart-seq2 protocol (n = 12, 24, and 10) and the protocol of “Smart-3SEQ starting from FFPE tissue on Arcturus LCM” (n = 12), from single granulosa cells in formalin-fixed ovarian sections, were also shown. (G) Boxplots of the number of detectable genes (estimated ≥1 copy expression) in single oocytes (red) and granulosa cells (blue) (n = 12 and 24, respectively) from the formalin-fixed ovarian sections, analyzed with DRaqL-Protease-Smart-seq2. (H) Frequency plots of gene expression levels in single oocytes and granulosa cells from the formalin-fixed sections, analyzed with DRaqL-Protease-Smart-seq2. (I) Bioanalyzer analysis of cDNA amplified with Smart-seq2 (from 10-pg total RNA), DRaqL-SMART-seq v4 and DRaqL-Smart-seq2 (from alcohol-fixed granulosa cells), and DRaqL-Protease-Smart-seq2 (from formalin-fixed granulosa cells).

Article Snippet: First, we examined the use of DRaqL in conjunction with a SMART-Seq v4 3′DE Kit (Takara Bio), a commercially available Smart-seq2-based kit that allows multiplex cDNA library preparation by including index sequences in the reverse transcription primers.

Techniques: Concentration Assay, Lysis, Amplification, Real-time Polymerase Chain Reaction, Isolation, Blocking Assay, Laser Capture Microdissection, Expressing