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| Status |
Public on Dec 31, 2019 |
| Title |
DBA/2J sperm cells pacbio |
| Sample type |
SRA |
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| Source name |
DBA/2J sperm cells
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| Organism |
Mus musculus |
| Characteristics |
strain: DBA/2J
cell type: sperm cells
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| Growth protocol |
Mice were maintained in an Assessment and Accreditation of Laboratory Animal Care credited specific pathogen free facility under a 12 h light, 12 h dark cycle.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Sperm were collected from the epididymis of DBA/2N males. MII oocytes were derived from 3 to 5 week-old C57BL/6J females induced to superovulate by injection of 7.5IU pregnant mare serum gonadotropin (PMSG, San Sheng), followed by injection of 7.5IU human chorionic gonadotropin (hCG, San Sheng) 48h later. Embryos were collected from the oviduct of the superovulated females after mating them with DBA/2N males. Oocytes and embryos were isolated in M2 medium (Sigma) at 20h post-hCG, then while oocytes were lyse directly, the embryos were not lyse until they develop to defined stages: zygotes (24-26h post hCG), two-cell stage (46-48h post-hCG), four-cell stage (54-58h post-hCG), eight-cell stage (68-70h post-hCG), blastocyst stage (94-96h post-hCG), with culturing in KSOM media (Merck Millipore) at 37℃ with 5% CO2.
Embryos were lysed in 50 μl of guanidine isothiocyanate solution (Invitrogen, 15577-018) at 42°C for 15 min. Nuclease-free water was added to 200 μl, and 3 volumes of absolute ethanol, 1/10 volume of 3 M acetate sodium (Invitrogen, AM9740) and 2 μl of glycogen (Roche, 10901393001) were added and mixed uniformly. After treatment at -80 °C for 2 h, total RNA was pelleted by centrifugation at 12,000g for 40 min. Total RNA pellets were dissolved in lysis solution and used as template for the first cDNA synthesis, cDNA was amplified by Smart-seq2 protocol1.
2 μg of each amplified cDNA is was transformed into SMRTbell templates using the PacBio SMRTbell Express Template Prep Kit. The libraries were sequenced using the PacBio Sequel System in Berry Genomics Company (Beijing, China).The sequencing libraries were constructed using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme Biotech, TD502-01) according to the manufacturer’s instruction. All libraries were sequenced on Illumina NovaSeq 6000 platform with a 150-bp paired-end module in Berry Genomics Company (Beijing, China).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Sequel |
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| Data processing |
Sequencing data were mapped mm10 reference genome using GMAP and STAR seperately
transcriptome of each pacbio sample were assembled by cufflinks
TPM of transcript were calculated using salmon
Genome_build: mm10
Supplementary_files_format_and_content: gtf file for pacbio sequencing data; plain txt file for illumina RNA-seq data, each file contain 2 column, the first column represents the transcript id and tpm
Supplementary_files_format_and_content: We do not have quantitative processed data for Pacbio data. The TPM results are calculated using gff generated from Pacbio data as references.
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| Submission date |
Oct 11, 2019 |
| Last update date |
Jan 01, 2020 |
| Contact name |
Wenjie Shu |
| E-mail(s) |
shuwj@bmi.ac.cn
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| Organization name |
Beijing Institute of Radiation Medicine
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| Street address |
Taiping Road 27
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| City |
Beijing |
| ZIP/Postal code |
100850 |
| Country |
China |
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| Platform ID |
GPL24198 |
| Series (1) |
| GSE138760 |
High resolution and allelic-specific annotation of preimplantation embryo transcriptome using long-read sequencing |
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| Relations |
| BioSample |
SAMN13018497 |
| SRA |
SRX6980457 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4118745_sperm.collapsed.filtered.gff.gz |
496.0 Kb |
(ftp)(http) |
GFF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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