GSM7947314: E319, 90-99 μm, replicate 2, scRNAseq; Bos taurus; RNA-Seq
PCR tubes containing single oocyte samples snap-frozen in PBS + RLT-Plus Buffer at the collection day were transported in dry ice to the Babraham Institute, UK. Oocyte lysates were thawed and transferred to a 96-well plate and incubated with Dynabeads (MyOne Streptavidin C1, Life Technologies) annealed to Smart-seq2 oligo-dTs 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) and reverse transcription was performed to produce cDNA. cDNA was amplified using KAPA HiFi HotStart ReadyMix (Roche), purified with Ampure XP beads with a 1:0.9 ratio, and eluted into 20 μL of water. 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. A total of 179 oocytes were 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.