title: GSM7112214: GV oocyte replicate 1; Mus musculus; RNA-Seq
library_construction_protocol: For each stage and replicate, total RNA was isolated from ~400 oocytes or embryos with Trizol reagent (Fisher Scientific, 15-596-018), followed by ethanol precipitation and RNA Clean & Concentrator-5 kit (Zymo Research, R1014) as per manufacturer's protocol. RNA was eluted in 7 ul nuclease-free water and used immediately for Nanopore PCR-cDNA sequencing library prep. Approximately 400 oocytes or embryos per biological replicate per stage were used per sequencing run. An identical amount of ERCC spike-in standards51 per cell was added to samples at the beginning of RNA extraction. Nanopore PCR-cDNA sequencing (PCS) was performed with an early access cDNA-PCR Sequencing Kit (PCS-110) as per manufacturer's protocol (Fig. S1A). This kit is most similar to the currently available cDNA-PCR Sequencing Kit (PCS-111). Briefly, ligation of Nanopore's proprietary cDNA-RT adapter (CRTA) to the 3' end of polyadenylated mRNAs enriched for transcripts with a minimum polyA tail length of 10 nt. Following enzymatic digestion of the adapter, RT with strand switching was performed, followed by selection of full-length transcripts by PCR amplification for 18 cycles. Sequencing adapters are then attached, followed by loading of the library onto a R9.4.1 flow cell (FLO-MIN106D) and sequenced in a Minion sequencing device with Minknow software (version 22.05.5). Libraries comprised of polyadenylated cDNA standards were prepared by PCR amplification using the following barcoded PCR primers from the Nanopore PCR-cDNA Barcoding Kit (SQK-PCB109), followed by attachment of sequencing adapters from the PCS-110 kit and sequencing on the Minion as per the PCS-110 protocol: pA_cDNA_standard_10: BC01, pA_cDNA_standard_40: BC02, pA_cDNA_standard_50: BC03, pA_cDNA_standard_70: BC04, pA_cDNA_standard_100: BC05, pA_cDNA_standard_130: BC06, and pA_cDNA_standard_150: BC07.