Transcriptome profiling of cytosol, endoplasmic reticulum (ER), and pellet fraction from the Xenopus laevis oocyte and mature egg

This genome-wide transcriptome analysis was aimed to reveal dynamic regulation of mRNA-ER association during Xenopus oocyte maturation. We extracted RNAs from the cytosolic, ER, and the pellet fraction of oocytes and eggs, respectively, and performed RNA-sequencing. These results showed that mRNA-ER association is down-regulated during oocyte maturation. Overall design: Three biological replicates were assigned for each group. Before RNA extraction, a mixture of spike-in RNAs (egfp, mCherry, mRuby2, firefly, renilla) was added to each fraction. Total RNA was extracted from the cytosol, ER, and pellet fraction in the four oocytes or four mature eggs using TRIzol reagent. Ribosomal RNA was removed with the Ribozero HMR kit (Illumina), and the RNA libraries were prepared using the TruSeq Stranded mRNAseq Sample Prep kit (Illumina). The libraries were quantitated by qPCR and sequenced on one lane for 101 cycles from each end of the fragments on a NovaSeq 6000 using a NovaSeq S1reagent kit. Fastq files were generated and demultiplexed with the bcl2fastq v2.20 Conversion Software (Illumina). Transcript abundance (TPM) was quantified by Kallisto, and the TPM of each RNA-seq data was converted to absolute attomolar concentration (aM) through the regression estimation with five spike-in RNAs.