Nextera xt tn5 transposase

Mouse subcutaneous adipose tissue, hypothalamus, and ovary were homogenized in 1 ml TRI reagent (T9424, Sigma-Aldrich). Total RNA was extracted as per the manufacturer’s instructions and quality was confirmed by Agilent 2100 bioanalyzer with RIN 6–7. Then 1 ng of total RNA was applied to bulk RNA-sequencing library. Sequencing libraries were generated according to Smart-seq3 protocol^{61 (link)}. Briefly, polyA(+) RNA was reverse transcribed by Maxima H-minus reverse transcriptase (Thermo Fisher). The second-strand synthesis was conducted by a template-switching reaction and 12 cycle of PCR was performed for cDNA amplification by KAPA HIFI HotStart polymerase (Roche). Then cDNA was purified by 22% PEG (Sigma-Aldrich) beads. Aglient 2100 BioAnalyzer (Agilent Technologies) was performed to check the quality and quantity of cDNA libraries. Sequencing libraries were generated by tagmenting 200 pg cDNA using Nextera XT Tn5 transposase (Illumina) and amplified for 10 cycles.

Single-MII oocytes were prepared by Smart-seq3 protocol. Following cell lysis, polyA(+) RNA was reverse transcribed by Maxima H-minus reverse transcriptase (Thermo Fisher) as mentioned below. cDNA amplification was performed by PCR (14 cycles) followed by beads purification. Sequencing libraries were generated by Nextera XT Tn5 transposase (Illumina) and amplified for 10 cycles.