|
| Status |
Public on Feb 14, 2020 |
| Title |
1GV_18c_2 |
| Sample type |
SRA |
| |
|
| Source name |
1GV_18c_single mouse oocyte
|
| Organism |
Mus musculus |
| Characteristics |
Stage: germinal vesicle (GV)
tissue: oocyte
amplification cycles: 18
|
| Growth protocol |
Mouse GV oocytes are cultured ex vivo in M2 medium.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
polyA RNAs: Each oocyte was lysed in RLT-plus buffer. The oligo-dT beads were used to isolate the polyA RNA. The oocyte fractions were picked after oocyte lysis
For polyA RNAs: Single oocyte RNA-seq libraries were prepared according to a published single-cell RNA seq pipeline with subtle modifications (Macaulay et al., 2016). Briefly, the oocytes at desired stages were collected individually into 2.5 ml RLT plus and stocked at -80 °C until all samples were collected. In the beginning of RNA purification, ERCC RNA spike-in mix (ThermoFisher 4456740) was diluted 10^5 fold, and 1 ml of the diluted ERCC mix was added to each lysis. Poly(A) RNA were isolated by oligo (dT) beads, reverse transcribed, amplified and purified. The purified cDNAs were analyzed by Bioanalyzer 2100 to confirm successful amplification range and quality. cDNAs passing a quality test were further constructed into sequencing libraries by Nextera DNA Sample Preparation Kit.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
ERCC added
1GV_18c_2_S1_L001
|
| Data processing |
Raw sequence reads were trimmed with Cutadapt 2.5 to remove adapters while performing light quality trimming with parameters "-m 10 -q 20, 20".
Sequencing library quality was assessed with FastQC v0.11.8 with default parameters. Trimmed reads were mapped to the Mus musculus mm10 reference genome plus ERCC.fasta using STAR v2.7.2a.
Multi-mapping reads were filtered using samtools 1.9. Uniquely aligned reads were then mapped to gene features using HTseq v0.9.1 as an unstranded library with default parameters.
A gene/ERCC count was considered valid when present in at least 5 reads in at least 2 libraries. Differential expression between groups of samples was tested using R version 3.5.1 (2018-07-02) with DESeq2 v1.24.0. The oocyte libraries were normalized by ERCC counts by defining the ERCC genes as the controlGenes when estimating the sizeFactors in DESeq2.
Genome_build: mm10
Supplementary_files_format_and_content: tab deliminated original counts of each gene
|
| |
|
| Submission date |
Feb 13, 2020 |
| Last update date |
Feb 15, 2020 |
| Contact name |
Di Wu |
| E-mail(s) |
[email protected]
|
| Organization name |
HHMI Janelia
|
| Department |
4DCP
|
| Street address |
19700 Helix Drive
|
| City |
Ashburn |
| State/province |
Virginia |
| ZIP/Postal code |
20147 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE145283 |
Oocytes, a single cell and a tissue |
|
| Relations |
| BioSample |
SAMN14100750 |
| SRA |
SRX7723728 |
