title: GSM8514528: phthmix; Mus musculus; RNA-Seq
library_construction_protocol: Following culture, follicles were immediately dissociated into a single cell suspension for single cell sequencing. Follicles from each treatment group were shredded using watchmaker forceps in a dissociation solution composed of DMEM/F12 (Thermo Fisher Scientific) containing 20 mg/mL of collagenase II (Thermo Fisher Scientific) and 50 U/mL DNAse 1 (Thermo Fisher Scientific). Cells were pipette mixed, incubated at 37 °C for 40 minutes, and then pipette mixed again. After centrifugation and removal of the supernatant, 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher Scientific) was added to the cells, pipette mixed, and incubated for 5 minutes. Cells were washed with cell culture media and strained through a 100 μm cell strainer. Cells were counted and concentration adjusted to 700 cells/μl for sequencing. Single-cell 3 cDNA libraries were prepared at the DNA Services laboratory of the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign. Single-cell suspensions were delivered to the facility and were counted and checked for viability >80% using the Nexcelom K2 brightfield/dual florescence cell counter (Nexcelom Biosciences, Lawrence MA) with AO/PI staining, then washed with PBS buffer containing 2.0% BSA, then recounted for library preparation. The cells were individually labeled with the 10x Cell Plex Multiplexing Oligo labels, washed, and pooled per the manufacturers instructions. The target number of labeled cells (30,000, to target 5,000 from each population) were converted into a single barcoded cDNA library with the Single-Cell 3 NextGEM v3.1 Chromium kit from 10X Genomics (Pleasanton, CA) following the manufacturers protocols. Following ds-cDNA synthesis, an individually-barcoded dual-index library compatible with the Illumina chemistry was constructed in parallel with the CellPlex library. The final libraries were quantitated on Qubit (Life Technologies, Grand Island, NY) and the average size determined on the AATI Fragment Analyzer (Agilent Technologies, Santa Clara, CA). The final libraries were then diluted to 5nM final concentration and further quantitated by qPCR on a BioRad CFX Connect Real-Time System (Bio-Rad Laboratories, Inc. CA), then pooled for sequencing. The final 10x single cell pool was sequenced on the Illumina NovaSeq 6000, using one lane of an S4 flowcell, as paired-reads with 150nt in length. The first read of the single cell library is used for the UMI and 10x barcode only, the 2nd read contains the RNA sequencing information. Basecalling and demultiplexing of raw data was done with the mkfastq command of the software Cell Ranger 6.0.1 (10x Genomics). Sorted data was posted to a password-secured AWS site for download and downstream processing.