ABSTRACT: Purpose: Investigate the transcriptomes of Tex14 heterogygous sample (with intercellular bridges intact) compared to Tex14 homozygous mutant (lacking intracellular bridges) to ascertain what effects the loss of cytoplasmic sharing has on mitotic to meiotic mouse ovary Methods: Single cell RNA sequencing libaries were created from embryonic day 13.5 mouse ovaries from Tex14 +/- and Tex14 -/- samples. Three Tex14+/- and two Tex14-/- E13.5 ovaries were pooled. Libaries were creating using 10X Chromium Single Cell 3' (v2 Chemistry) library kits per 10X posted protocols. Samples were sequenced on HiSeq 4000 (Illumina) with paired-end sequencing parameters: Read1, 98 cycles; Index1, 14 cycles; Index2, 8 cycles; and Read2, 10 cycles. Tex14+/- sample had 23,567 mean reads per cell, and Tex14-/- had 19,735 mean reads per cell. Since 10X Genomics recommends an average of 60k raw reads per cell, we submitted our two libaries for deeper sequencing. Our second sequencing run was done on a Novaseq S2 flowcell with the following parameters: Read 1 Length 26bp; Index 1 Length 8bp; Index 2 Length 0bp; Read 2 Length: 98 bp. Follwing deeper sequencing, Tex14+/- sample had 55,744 mean reads per cell, and Tex14-/- had 41,117 mean reads per cell. Cells which passed quality control metrics were further analyzed. Results: We used CellRanger v2.1.0 to align the sequenced data to the 10X Genomics pre-built mm10 genome. We mapped about 300,000,000 sequence reads for each shallowly sequenced library, and 750-800,000,000 reads for the deeply sequenced libraries. We used Seurat v3 to keep high quality cells, excluding cells which did not all of the following metrics: genes expressed in at least 3 cells, fewer than 25,000 unique fragments (to exclude cell doublets), greater than 200 genes expressed (to exclude GEMS with no cells), and cells with 7.0% or less mitochondrial gene expression. After quality control exclusion, cell counts for the gene by cell matrices are as follows: tex14 het shallow, 12260 cells; tex14 ko shallow, 14715 cells; tex14 het deep, 11794 cells; tex14 ko deep, 13982 cells. Conclusions: Our study represents the first single cell RNA sequencing experiment conducted on fetal mouse ovaries lacking intracellular bridges. We observed that the absence of Tex14 did not alter the transcriptional composition of ovarian somatic cells, but specifically attenuated the level of pluripotency-associated transcripts as germ cells enter meiosis.
