Kapa hotstart mouse genotyping kit

The Math1-GFP line, J2XnGFP(Math1-nGFP tg) mice, were a gift from Jane E. Johnson, UT Southwestern^{9 (link)} and maintained on a C57BL/ 6 background. Rosa26-ZO1-EGFP mice were obtained from RIKEN Laboratory^{13 (link)} (accession no. CDB0260K) and maintained on a C57BL/6 background. All animal procedures were approved by the Animal Care and Use Committee at Tel Aviv University (04-16-014). Genotyping was performed using the KAPA HotStart Mouse Genotyping Kit (Sigma, KK7352) using GFP primers listed in Supplementary Table 2.

Expression of OVA on FRCs from B6.iFABP-tOVA mice was assessed after FACS of mLN FRCs from B6.WT and B6.iFABP-tOVA into lysis buffer (purity > 95%). RNA was isolated using RNeasy Mini Kit (Qiagen), quantified and cDNA synthesized with the RevertAid first strand cDNA synthesis kit (Thermo Fisher Scientific), and PCR was performed by the KAPA HotStart Mouse Genotyping Kit (Sigma-Aldrich) using the primer pairs: Gapdh, 5′-AGTATGACTCCACTCACGGC-3′ plus 5′-ATGTTAGTGGGGTCTCGCTC-3′; Ova, 5′-GCTGCAGATCAAGCCAGAGAGC-3′ plus 5′-ATTGATTTCTGCATGTGCTGC-3′.

Mice were used as approved by the Cleveland Clinic and Children’s Hospital of Philadelphia Institutional Animal Care and Use Committees. Mice carried Sox4, Sox11, and Sox12 conditional null alleles^{18 (link),105 (link)} and a Prx1Cre^{106 (link)}, OsxCre^{107 (link)} or Col10Cre^{108 (link)} transgene or an Acan^CreERT2 allele^{109 (link)}. Mice with R26^SOX4 alleles were generated as described below. Mice were genotyped by PCR of genomic DNA using KAPA HotStart Mouse Genotyping Kit (HSMGTKB, Sigma–Aldrich) and primers as listed (Supplementary Table 1). Unless otherwise stated in Results, mice featuring OsxCre were fed with doxycycline-containing food (200 mg doxycycline/kg food; S3888, Bio-Serv) to prevent Cre expression. Cre recombinase activity was induced in 21-day-old Acan^CreERT2/+ mice by intraperitoneal injection of tamoxifen (1 mg/10 g body weight; T5648, Sigma–Aldrich) for 4 consecutive days. Bilateral ovariectomy was performed upon mouse anesthesia by making a single midline incision on the dorsal surface, ligating the uterine horns, and removing the ovaries^{110 (link)}. Success of the procedure was assessed postmortem by the absence of ovaries and reduction in uterus size.

All animal procedures were approved by the Animal Care and Use Committee (IACUC) at Tel Aviv University (01-17-101) and performed according to the NIH Guide for the Care and Use of Laboratory Animals. For every experiment, the age is specified in the figure legend. Mice were maintained on a C57Bl/6J background. Genotyping was performed on DNA prepared from ear punch biopsies, and extracted and amplified using the KAPA HotStartMouse Genotyping Kit (Sigma, KK7352). Genotyping primers for the WT allele were WT_FWD (5-ACT​CCC​AGC​TCC​AAG​CTA​CA-3) and WT_REV (5-GCA​GAG​CCA​AAG​AAA​CCA​AG-3), and for the galactosidase gene were LacZ_FWD (5-GTC​TCG​TTG​CTG​CAT​AAA​CC-3) and LacZ_REV (5-TCG​TCT​GCT​CAT​CCA​TGA​CC-3). Cycling conditions were an initial 3-min denaturation at 95°C followed by 35 cycles of 30 s 95°C, 30 s 60°C, and 30 s at 72°C, with a final elongation of 3 min at 72°C. PCR products were loaded into a 2% agarose ethidium-bromide gel for electrophoresis.

All animal work was performed in the Core Animal Facility at the University of South Australia, with approvals from the institutional animal ethics committee (AEC numbers U23‐16, U06‐19, U25‐19, U41‐19) and the institutional biosafety committee (IBC0090 and IBC0143). The animal facilities were operated according to international animal welfare rules (Federation for Laboratory Animal Science Associations guidelines and recommendations). Mouse colonies were maintained in specific pathogen‐free conditions in individually vented cages with 12–12 h light‐dark cycle. The following mouse strains were used in this study: Arrdc4^–/– mice (Mackenzie et al., 2016); C57BL/6J females and CBA/CaH males were used to generate CBAB6F1 female progeny used in the in vitro fertilization experiments. Male Arrdc4^–/– mice used for experiments were between 4 and 6 months of age, male CBA/CaH males and C57BL/6J females used for breeding were 2–12 months of age, and B6CBAF1 females used for in vitro fertilization studies were 3–7 weeks of age. Average litter size was calculated by counting the number of pups at birth (Table 1).
Genotyping was carried out on tail or ear clippings from Arrdc4^–/‐ mice using the KAPA HotStart Mouse Genotyping Kit (Sigma) using the genotyping primers listed in Supplemental Table S1, or by the Transnetyx Automated Genotyping Services (Cordova, TN).