GSM5665255: F-TAF4b, rep 1; Mus musculus; OTHER
Mice that were homozygous for an Oct4-EGFP transgene (The Jackson Laboratory: B6;129S4-Pou5f1tm2Jae/J) were mated for germ cell 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. For library preparation, the KAPA HyperPrep kit (Roche Cat. No 07962363001) was used with New England Biolabs NEBNext Multiplex Oligos for Illumina (NEB #E7335). After library amplification through PCR, libraries were size selected through gel extraction (~150-650 bp) and cleaned up using the Qiagen QIAquick Gel Extraction Kit (Cat. # 28704). CUT&RUN libraries in EB buffer were shipped to GENEWIZ (GENEWIZ Inc., NJ) on dry ice. Sample QC, sequencing, and initial bioinformatics were done at GENEWIZ. CUT&RUN