GSM8898592: High-fat diet,Transcriptomics,MII-2; Mus musculus; OTHER
We collected GV and MII stage oocytes (n=4 per sample) from ND and HFD groups, lysing them in 10 µL ice-cold sample buffer (Vazyme N711 containing 0.2 µL 1:2500 ERCC spike-in [Invitrogen 4456740] and RNase inhibitor) for 10 minutes, then divided each lysate into 2.0 µL for transcriptomics and 8.0 µL for proteomics. For ribosome profiling, we functionalized RiboLace beads (Immagina RL001), resuspended them in the 8 µL lysate supplemented with binding buffer (100 µg/mL cycloheximide, 1 mM DTT, 5 mM MgCl₂, 150 mM NaCl, 20 mM Tris-HCl pH 8.0) and RNase inhibitor, then incubated at 4°C for 1 hour with 3 rpm rotation. After magnetic rack washing with W-buffer, we eluted ribosome-bound RNA in 12 µL RLT buffer (Qiagen 74004) containing β-mercaptoethanol and GlycoBlue, then purified using VAHTS RNA Clean Beads (Vazyme N411) with 2M LiCl precipitation following manufacturer protocols. For ultra-sensitive T&T-seq library construction, total RNA (2 µL lysate) and ribosome-bound full-length RNA were reverse transcribed into cDNA and amplified for 18 PCR cycles using the Single Cell Full Length mRNA-Amplification Kit (Vazyme N711), followed by purification with VAHTS DNA Clean Beads (Vazyme N411), with cDNA concentration measured by Qubit (Invitrogen) and size distribution (~2000 bp) analyzed using Bioanalyzer 2100 (Agilent), then index libraries were prepared using TruePrep DNA Library Prep Kit V2 (Vazyme TD502) and size-selected (250-450 bp) with VAHTS DNA Clean Beads before quantification and paired-end sequencing (PE150) on Illumina Novaseq 6000 (Azenta Life Sciences). For RNA-seq library preparation, GV and MII stage oocytes (n=4/sample) from ND/HFD groups were lysed in sample buffer (Vazyme N711) containing 0.2 µL 1:2500 ERCC spike-in (Invitrogen 4456740), followed by cDNA synthesis using Oligo dT primers and Discover-sc reverse transcriptase for template switching adapter addition, PCR amplification of full-length cDNA, magnetic bead purification (Vazyme N411), library construction using TruePrep Kit V2 (Vazyme TD503) with 15 PCR cycles, size selection (250-450 bp), Qubit quantification, Bioanalyzer quality assessment, and PE150 sequencing on Illumina Novaseq 6000 (Azenta Life Sciences).