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Sample GSM7112222

Query DataSets for GSM7112222

Status

Public on Mar 14, 2024

Title

L2 oocyte replicate 1

Sample type

SRA

Source name

L2 oocyte

Organism

Mus musculus

Characteristics

cell type: L2 oocyte

Growth protocol

Mouse oocytes were isolated from C57B6J female mice. For GV oocytes, 4-week old females were stimulated by intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (PMSG; Lee BioSolutions, 493-10), ovaries were harvested 48 hours after PMSG injection, and denuded oocytes collected after brief digestion with trypsin (Fisher Scientific, 25200056), collagenase (Sigma-Aldrich, C9407), and DNAse in M2 media (Sigma-Aldrich, M7167). For MII oocytes, mice were injected with 5 IU HCG (Sigma-Aldrich, C1063) 48 hours after PMSG induction and ovulated MII oocytes collected from the ampulla 15 hours post-HCG and cumulus cells by brief hyaluronidase (Sigma-Aldrich, H4272) digestion per standard protocol. Mouse embryos were isolated from B6D2F1/J female mice mated with C57B6J males of known fertility at the time of hCG injection. 1-cell, early 2-cell, and late 2-cell embryos were collected at 27-28, 30-32, and 46-48 hrs post-HCG.

Extracted molecule

total RNA

Extraction 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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

MinION

Description

processed data file header: L2_r1

Data processing

Raw fast5 files were basecalled with Guppy (version 6.0.6) following parameters: --config dna_r9.4.1_450bps_hac.cfg --fast5_out --trim_strategy none --records_per_fastq 0 --device cuda:all:100% --recursive. and full length reads with all adapter sequences (from the CRTA, PCR primers, and sequencing adapter) were filtered by Pychopper (version 2.5.0) following parameters: -k PCS110. Only full-length reads with a minimum basecalling Q-value score of 9 and all adapter sequences were retained for further analysis. Reads were mapped to the GRCm38 mouse transcriptome with minimap (version 2.17) using the following parameters: -a -x map-ont. Reads per transcript were quantified with Salmon (version 0.14.1) using the following parameters: salmon quant -l A --noErrorModel. Read counts were normalized with ERCC standards by dividing read counts for each gene by the mean read count amongst ERCCs. Differential expression analysis was performed with DESeq2 (version 1.34.0).
Assembly: GRCm38
Supplementary files format and content: Tab-delimited text file includes normalized read counts exported from DESeq2 for each sample.

Submission date

Mar 22, 2023

Last update date

Mar 14, 2024

Contact name

Heidi Cook-Andersen

E-mail(s)

[email protected]

Organization name

University of California, San Diego

Department

Department of Reproductive Medicine