GSM5292819: E14.5, Taf4b-Def rep2; Mus musculus; RNA-Seq
Mice that were homozygous for an Oct4-EGFP transgene (The Jackson Laboratory: B6;129S4-Pou5f1tm2Jae/J) and heterozygous for the Taf4b-deficiency mutation were mated for mRNA collections. Timed matings were estimated to begin at day 0.5 by evidence of a copulatory plug. To dissociate gonadal tissue into a single-cell suspension, embryonic gonads were harvested and placed in 0.25% Trypsin/EDTA and incubated at 37°C for 25 minutes, as previously described (Gura et al., 2020). Eppendorf tubes were flicked to dissociate tissue halfway through and again at the end of the incubation. Trypsin was neutralized with FBS. Cells were pelleted at 1,500 RPM for 5 minutes, the supernatant was removed, and cells were resuspended in 100 μL PBS. The cell suspension was strained through a 35 μm mesh cap into a FACS tube (Gibco REF # 352235). Propidium iodide (1:500) was added to the cell suspension as a live/dead distinguishing stain. Fluorescence-activated cell sorting (FACS) was performed using a Becton Dickinson FACSAria III in the Flow Cytometry and Cell Sorting Core Facility at Brown University. A negative control of a non-transgenic mouse gonad was used for each experiment to establish an appropriate GFP signal baseline. Dead cells were discarded and then cells were sorted at 4°C in PBS based on GFP signal into GFP+ or GFP- samples. These cells were then spun down at 1,500 RPM for 5 minutes, had PBS removed, and were then resuspended in Trizol (ThermoFisher # 1556026). If samples had roughly less than 50 µL of PBS in the tube, Trizol was added immediately. Samples were stored at -80°C. Embryonic germ cells in Trizol were shipped to GENEWIZ (GENEWIZ Inc., NJ) on dry ice. Sample RNA extraction, sample QC, library preparation, sequencing, and initial bioinformatics were done at GENEWIZ. RNA was extracted following the Trizol Reagent User Guide (Thermo Fisher Scientific). Glycogen was added (1 µL, 10 mg/mL) to the supernatant to increase RNA recovery. RNA was quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with TapeStation (Agilent Technologies, Palo Alto, CA, USA) to see if the concentration met the requirements. SMART-Seq v4 Ultra Low Input Kit for Sequencing was used for full-length cDNA synthesis and amplification (Clontech, Mountain View, CA), and Illumina Nextera XT library was used for sequencing library preparation. The sequencing libraries were multiplexed and clustered on a lane of a flowcell. After clustering, the flowcell was loaded onto an Illumina HiSeq 4000 according to manufacturer's instructions. The samples were sequenced using a 2x150 Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS) on the HiSeq instrument.  Raw sequence data (.bcl files) generated from Illumina HiSeq were converted into fastq files and de-multiplexed using bcl2fastq (v. 2.17). One mismatch was allowed for index sequence identification.