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Sample GSM6260526

Query DataSets for GSM6260526

Status

Public on Jun 29, 2022

Title

Oocyte, transitioning to primary, rep1

Sample type

SRA

Source name

Oocyte

Organism

Mus musculus

Characteristics

cell type: Oocyte

Extracted molecule

total RNA

Extraction protocol

Follicles were incubated in Accutase solution (Stemcell Technologies, Canada) (Zhang et al., 2018) until somatic cells partially detached from each other. Specifically, primordial and transitioning follicles were incubated in Accutase solution for 5 minutes, and primary and secondary follicles were incubated in Accutase solution for 10 minutes on 37 °C heated stage following agitation to dissociate somatic cells from the oocyte. Primordial and transitioning follicles were pipetted with 20 μm Pasteur pipet prepared by a micropipette puller (Sutter Instrument, CA, USA), primary follicle with a 30 μm Pasteur pipet and secondary follicle with a 50 μm stripper tip (Origio Inc.). Single cells were washed in three drops of 50 μL L-15/PVA with 100 μL/mL FBS.
scRNA-seq libraries were generated using SMART-Seq2 protocol (Trombetta et al., 2014). Briefly, cDNA was reversed transcribed from single cells using Maxima RT (Thermo Fisher Scientific) and whole transcriptome amplification (WTA) was performed. WTA products were purified using the Agencourt AMPure XP beads (Beckman Coulter, IN, USA) and used to prepare paired-end libraries with Nextera XT (Illumina, CA, USA).

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

Smart-seq2

Data processing

Raw sequencing data were demultiplexed and aligned to the GRCm38 genome using publicly available scripts on Terra (github.com/broadinstitute/TAG-public)
Cells expressing less than 500 genes were excluded from further analysis and genes were filtered to contain only those that were protein coding.
To filter for high-quality cells for each category, we utilized the AddModuleScore function in Seurat v4 (Hao et al., 2021) for scoring marker gene expression modules established from published datasets (Wang et al., 2020, Tharp et al., 2020, Zhang et al., 2018, Pangas et al., 2006) (Supplementary Table 1). Oocytes that scored highly for somatic cell gene expression (>2 standard deviations above the mean somatic cell module score) were removed. Similarly, somatic cells that scored highly for oocyte gene expression were also excluded.
Assembly: GRCm38/mm10
Supplementary files format and content: Tab-separated counts and tpm files

Submission date

Jun 22, 2022

Last update date

Jul 02, 2022

Contact name

Daniela Dolores Russo

E-mail(s)

ddrusso@mit.edu

Organization name

Ragon Institute of MGH, MIT and Harvard

Lab

Shalek Lab