GSM9312203: Nr5a2_8C_Klf5_KD_TIPseq_rep1; Mus musculus; OTHER
TIP-seq was performed according to the original protocol (Bartlett et al., 2021). Embryos with intact zona pellucida were incubated with ice-cold extraction buffer (25 mM HEPES-NaOH (pH 7.4), 50 mM NaCl, 3 mM MgCl2, 300 mM sucrose, and 0.5% Triton X-100) on ice for 6-7 min and were washed three times with ice-cold extraction buffer without Triton X-100. Pre-extracted embryos were immediately lightly fixed in Dulbecco's phosphate-buffered saline (DPBS) with 0.1% formaldehyde for 2 min at room temperature. Embryos were incubated in antibody buffer (20 mM HEPES-NaOH (pH7.5), 150 mM NaCl, 0.5 mM Spermidine, 0.1% BSA, 2 mM EDTA and 1x Protease inhibitor cocktail (Roche) with primary antibody (1:100 dilution, NR5A2 (R&D Systems, PP-H2325-00)) overnight at 4°C on nutator. Embryos were washed three times through antibody buffer and further incubated with secondary antibody (1:100 dilution, rabbit anti-mouse IgG H&L (ab46540, abcam)) for 1.5 hours at room temperature. Cells were washed three times with Wash buffer (20 mM HEPES-NaOH (pH 7.5), 150 mM NaCl, 0.5 mM Spermidine, and 1x Protease inhibitor cocktail (Roche)) and further incubated with homemade pA-Tn5 or pAG-Tn5 adaptor complex (1:250 dilution) for 1.5 hours at room temperature. Embryos were washed three times through Wash-300 buffer (20 mM HEPES-NaOH (pH 7.5), 300 mM NaCl, 0.5 mM Spermidine, and 1x Protease inhibitor cocktail (Roche)). Embryos were transferred into a Axygen 0.2 mL Maxymum Recovery Thin Wall PCR Tubes with 50 µl of Tagmentation buffer (10 mM MgCl2 in Wash-300) and incubated at 37°C for 1 hour. The samples were then deproteinized and reverse-crosslinked by adding 1.7 μl of 0.5 M EDTA, 1 μl of 10% SDS (0.2% final) and 0.5 μl of 20 mg/ml proteinase K (Roche #03115887001) and incubating for 2 hours at 50°C, followed by overnight incubation at 65°C. After adding 2 μl of homemade spike-in Drosophila DNA, tagmented gDNA was purified using 2x volume of AMPure XP magnetic beads (Beckman Coulter), and DNA and beads were resuspended in 8 μl of RNase-free water. The further steps involving gap filling, in vitro transcription, cDNA synthesis and cDNA tagmentation were performed according to the original TIP-seq protocol (Bartlett et al., 2021). The tagmented DNA was amplified 50 µl of reaction with 1×Q5 High-Fidelity Master mix (NEB) and 0.25 µM barcoded primers using the following PCR program: 72 °C for 5 min; 98°C for 30 sec; 12 cycles of 98°C for 10 sec and 63°C for 10 sec; final extension at 72 °C for 1 min and hold at 4°C. After the PCR reaction, libraries were purified by 1.1×AMPure XP magnetic beads (Beckman Coulter) and were eluted in 20 µl of RNase-free water. Purified DNA libraries were sequenced on a NextSeq 500 (Illumina), Novaseq 6000 (Illumina), or AVITI (Element Biosciences) with paired-end reads.