title: Lepidochelys olivacea Input Gonad 33C stage25 stage26 b2t replicate1 ChIPseqinput
design: Urogenital complexes were removed from embryos incubated at 26C (male-producing temperature) or 33C (female-producing temperature). The gonads and mesonephros were separated and sliced in small pieces (~2 mm3) in 1.0 ml of MACS buffer (1.0 XPBS Ph 7.2, 2 mM EDTA, 0.5 BSA) and submitted to cell-disaggregation. 60 gonads at stages 25 and 26 were pooled. They were incubated in 5 mg collagenase type I (Gibco, #17018029), in 1.0 ml of buffer MACS, 5 min at 37C, resuspended twice for 5 min, and filtered through a sterile 70 m mesh nylon filter. Disaggregated cells were washed twice in MACS buffer at 4C, fixed with 1% paraformaldehyde in PBS for 15 min, washed in PBS containing 0.125 M Glycine (BD FACS) for 15 min at 4C, and centrifugated at 2000xG for 15 min at 4C. After that, the disaggregated cells were centrifugated and washed three times at 4C with PBS, frozen with dry ice, and kept at 70C until use. Samples were cross-linked with 1% formaldehyde for 10 min at room temperature, and cross-linking was stopped by adding 0.125M glycine. All steps were performed at 4C unless otherwise indicated. Cells were washed in PBS and resuspended in wash buffer (10 mM HEPES at pH 7.5, 10 mM EDTA, 0.5 mM EGTA, and 0.75% Triton X-100 and 1X protease inhibitor cocktail, PIC, Roche, #11697498001). Cells were lysed in swelling buffer (25 mM Tris at pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, 1% SDS, 0.5% sodium deoxycholate, PIC) for 30 min. Chromatin was sonicated to 150300 bp. We used the Bioruptor sonication device (Diagenode) and the 500-Watt Ultrasonic Processor device (Cole-Parmer); 10-15 cycles 30sON/30sOFF, 10-15 cycles 50sON/90sOFF, 10-15 cycles 50sON/90sOFF, and 7-12 cycles 30sON/60sOFF. Insoluble material was removed by centrifugation at 13,000 rpm for 30 min. Before Immunoprecipitation (IP), a spike-in chromatin standard corresponding to 3% of cross-linked and sonicated mouse liver chromatin was added to all samples as a control. Sonicated chromatin was incubated overnight with bovine serum albumin (BSA)-blocked magnetic Dynabeads Protein G (Invitrogen, #10003D) previously coupled with purified anti-JARID2 antibody (Abcam, #93288). We used five micrograms of primary antibody per 1 million cells. The magnetic beads were washed in three wash buffers; i) twice in wash buffer A (50 mM Tris at pH 8, 1 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% NP40, 0.5% sodium deoxycholate, PIC), ii) once in wash buffer B (50 mM Tris at pH 8, 1 mM EDTA, 500 mM NaCl, 0.1% SDS, 1% NP40, 0.5% sodium deoxycholate, PIC) and iii) once in wash buffer C (50 mM Tris at pH 8, 1 mM EDTA, 250 mM LiCl, 1% NP40, 0.5% sodium deoxycholate, PIC). Chromatin was eluted off magnetic beads in 1% SDS and 0.1 M NaHCO3 and cross-links were reversed in the presence of 1 mg/ml Proteinase K and 150 mg/ml RNase overnight at 65 C. DNA was purified using a Zymo Research ChIP DNA purification kit (#D5201) eluting in 50 L of water, and quantified with a Qubit Fluorometer and the dsDNA High Sensitivity Assay kit (Thermo Scientific). ChIP-seq libraries were prepared using the Illumina TruSeq DNA PCR-Free library prep kit. Libraries were sequenced on the Illumina NextSeq 500 platform (75 nucleotides, paired-end).