Magnetic rack

Prior to the immunoprecipitation protocol, internal methylated and unmethylated standards were added to each sample for evaluation of immunoprecipitation performance (Diagenode, Seraing, Belgium). Samples were heat-denatured and immediately placed in ice for 10 min. An aliquot of each sample was stored at − 20 °C as an input control. An amount of 10 μg of antibody (monoclonal mouse anti-5-methylcytidine. Diagenode, Seraing, Belgium) was added to each sample. The mixture was incubated on a rotating platform at 4 °C. Then to the DNA-antibody mixture, a volume of 50 μl of magnetic Dynabeads® Protein G (Invitrogen Life Technologies Corporation, Gaithersburg, MD, USA) was added. The samples were later incubated for 2 h on a rotating platform at 4 °C. A magnetic rack (Invitrogen Life Technologies Corporation, Gaithersburg, MD, USA) was used to remove the supernatant and for the washing steps. In total we performed three washes and later on we resuspended the magnetic beads in 250 μl of digestion buffer (50 mM Tris; pH 8.0, 10 mM EDTA and 0.5% SDS) with 100 μg of Proteinase K (Sigma Chemical Co., St. Louis, MO, USA). The digestion with Proteinase K was realized in a hybridization oven with rotation at 50 °C.

Nuclear fractions from 2 × 10⁷ transfected and infected 293T cells were lysed by 600 µl of prechilled NP-40 lysis buffer (50 mM HEPES-KOH at pH 7.4, 150 mM KCl, 2 mM EDTA, 0.5% [vol/vol] NP-40, 0.5 mM DTT, Roche complete EDTA-free protease inhibitor cocktail, 40 U/ml Promega RNasin RNase inhibitor) while incubated on ice for 5 min. Lysates were cleared by centrifugation (16,000 relative centrifugal force [RCF], 4°C, 15 min). A 200-µl aliquot of the supernatant was used to quantify the amount of input RNA. Another 200-µl aliquot of the supernatant was incubated with 20 µl protein G Dynabeads (Invitrogen) precoated with 1 µg of anti-myc tag antibody or the same amount of an IgG2a isotype control antibody (Southern Biotech) respectively. RNP binding was performed overnight at 4°C. Washing was done with prechilled immunoprecipitation (IP) wash buffer (50 mM HEPES-KOH at pH 7.4, 150 mM KCl, 2 mM EDTA, 0.05% [vol/vol] NP-40, 0.5 mM DTT, complete EDTA-free protease inhibitor cocktail, 40 U/ml Promega RNasin RNase inhibitor) for six times with the aid of a magnetic rack (Invitrogen). Proteins bound to the beads were recovered from 10% of the beads by heat denaturation and characterized by Western blot analyses. RNA associated with the remaining 90% of the beads was purified by the TRIzol method. Input RNA and bead-associated RNA were analyzed by RT-qPCR.

After 5 days in culture, the gonads were removed from the membrane dissociated in collagenase/dispase solution (10,269,638,001, Roche) at 37 °C, for 20 min. The cell suspension was incubated for 2 h in 12-well plates pre-treated with gelatin (G1890, Sigma) for somatic cell adhesion. The medium containing non-adhered cells (including PGC) was centrifuged and resuspended in 1X PBS (pH 7.2) containing magnetic beads pre-incubated with biotinilated Dolichus biflorus agglutinin (DBA). The suspension was incubated for 1 h at 4 °C rocking and placed in a magnetic rack (Invitrogen). The positive selected cells (PGC) and the negative selected cells (somatic cells) were submitted to RNA isolation using TRIzol® (Invitrogen). The cells that adhered to the 12-well plates in the first step of the selection were treated with 1X trypsin (59418C, Sigma) and combined with the magnetic negative selection cells for RNA isolation. The cDNA was obtained using the Superscript III Reverse Transcriptase (Invitrogen). Conventional PCR was performed using specific primers for the developmental genes Pax6 (FP 5′-agtgaatgggcggagttatg-3′ and RP 5′-aacaaccacatgagccaaca-3′) and Dazl (FP 5′-gaaatggcccacaaaagaaa-3′ and 5′-ttaagcactgcccgacttct −3′).

For ChIP experiment, Cells were fixed by cross-linking with a final concentration of 1% formaldehyde solution for 10 min at room temperature and then quenched with 125 mM glycine for 5 min. After washing with cold PBS containing a protease inhibitor cocktail and PMSF twice, these cells were lysed with cell lysis buffer and nuclear lysis buffer. The clarified lysate was subject to sonication. The chromatin was then sheared to fragments of 200–500 bp. The chromatin was used for ChIP incubating with Dynabeads protein G (Thermo Fisher Scientific) coated with either an anti-dMyc antibody (P4C4-B10; DSHB) or mouse IgG control antibody overnight at 4°C on a rotating platform. After repeated washes using a magnetic rack (Thermo Fisher Scientific), dMyc-bound genomic DNA was eluted from Dynabeads, and then the cross-links were reversed at 65°C for 4h (or overnight). DNA fragments then were purified with AxyPrep PCR Cleanup Kit (Axygen). qRT-PCR analysis was performed using the DNA from the Input and ChIP experiments with primers listed in S2 Table. At least three independent experiments were carried out for the miR-277 promoters, as well as for Fibrillarin gene served as a positive control [78 (link)].

Vacutainer tubes containing acid citrate dextrose (ACD) anticoagulant, anti-CD63 antibody-conjugated magnetic beads (catalog no.: 10606D), magnetic rack (catalog no.: 12321D), TaqMan^® miRNA ABC purification kit–Human Panel A, TaqMan^® microRNA reverse-transcription kit, MicroBCA^™ Protein Assay Kit, phosphate-buffered saline (PBS), RIPA buffer, and proteinase inhibitor were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Cel-miR-238-3p and miR-126-3p were purchased from Genomics (New Taipei City, Taiwan). Lyophilized bovine serum albumin (BSA) and prostaglandin E1 (PGE1) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Iodixanol (OptiPrep) was obtained from Axis-Shield (Dundee, Scotland, UK). Sodium cacodylate buffer and 300-mesh Formvar/carbon-coated grids were obtained from Electron Microscopy Sciences (Hatfield, PA, USA).

Polyadenylated RNA (0.036 pmol) encoding for HA-protein of interest was injected into stage VI oocytes, and 16-h post microinjection oocytes were lysed in 9 μL/oocyte cold IP lysis buffer (20 mM Tris–HCl pH 8, 100 mM NaCl, 0.4% NP40, 1 mM EDTA, 1 mM MgCl₂, 1 × Complete EDTA-free protease inhibitors (Roche)) and clarified by centrifugation. 1 × H1K phosphatase inhibitors (80 mM sodium β-glycerophosphate, 0.5 mM sodium orthovanadate) were added to the recovered aqueous phase. In IPs with RNase treatment, the lysates were incubated for 15 min at 37 °C with 0.1 μg/μL of RNase A, DNase, and protease-free (Thermo Fisher Scientific). One hundred fifty microliters of HA-conjugated magnetic beads (Pierce Anti-HA Magnetic Beads, Thermo Fisher Scientific) was used per mL of clarified lysate. Lysate and beads were then incubated for 2–20 h. The beads were washed thrice in one volume of lysis buffer. Purified proteins were eluted from the beads with Laemmli sample buffer without DTT. Supernatants were then recovered with a magnetic rack (Thermo Fisher Scientific), and beads were discarded and DTT was added to the supernantant at 180 mM final concentration. The eluates were then analyzed by Western blot (WB).

ChIP was performed as previously described [60 (link)]. Transfected S2 cells were cross-linked with 1% (v/v) formaldehyde for 10 min. They were then lysed with cell and nuclear lysis buffers. Clarified lysates were then sonicated. The chromatin was sheared into 200–800-bp fragments and used in ChIP incubation with Dynabeads protein G (Thermo Fisher Scientific, Waltham, MA, USA) coated either with anti-V5 antibody (ABclonal Biotechnology Co. Ltd., Wuhan, Hubei, China) or anti-mouse IgG antibody on a rotating platform at 4 °C overnight. After repeated washings on a magnetic rack (Thermo Fisher Scientific, Waltham, MA, USA), the V5-bound genomic DNA was eluted from the Dynabeads and the cross-links were reversed at 65 °C overnight. The DNA fragments were purified with an AxyPrep PCR cleanup kit (Axygen Scientific, Union City, CA, USA). The qPCR analysis was performed using DNA from the Input and ChIP experiments and the primers listed in Supplementary Table S3. At least three independent experiments were conducted on the AMPs promoters.