Transcriptome Analysis of Single Oocytes

RNA from individual oocytes was isolated using the Genome & Transcriptome protocol [27 (link)], and cDNA conversion, amplification, and purification were performed as described by [28 (link)]. Briefly, oocyte lysates were transferred to a 96-well plate and incubated with Dynabeads (MyOne Streptavidin C1, Life Technologies) annealed to Smart-seq2 oligo-dTs [29 (link), 30 (link)] to capture polyadenylated mRNA. The remaining lysate containing the DNA was transferred to a new 96 well-plate for a subsequent bisulphite conversion analysis. The beads containing the mRNA were diluted in reverse transcription master mix using SuperScript™ II Reverse Transcriptase (Invitrogen). cDNA was amplified (14 cycles) using KAPA HiFi HotStart ReadyMix (Roche), purified with Ampure XP beads with a 1:0.9 ratio, and eluted into 20 μL of water [29 (link), 30 (link)]. Amplified cDNA was quantified using a High Sensitivity DNA Assay chip (Agilent Technologies), and libraries were prepared using the Nextera XT Kit (Illumina) with ~ 300 pg of cDNA in two different replicates (Supplementary Fig. S1). The individual transcriptome of 179 oocytes was sequenced on the NextSeq500 HighOutput 150 bp Single End (replicate 1) and 75 bp Single End (replicate 2) with a sequence depth of ~ 2 million reads.