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| Status |
Public on May 14, 2019 |
| Title |
embryo1-P,1,P target site amplicon |
| Sample type |
SRA |
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| Source name |
E8.5 embryo
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| Organism |
Mus musculus |
| Characteristics |
developmental stage: E8.5
tissue: whole embryo
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| Treatment protocol |
To enable in vivo lineage tracing, B6D2F1 strain female mice (age 6 to 8 weeks, Jackson Labs) were superovulated by sequential intraperitoneal injection of Pregnant Mare Serum Gonadotropin (5IU per mouse, Prospec Protein Specialists) and Human Chorionic Gonadotropin (5IU, Millipore) 46 hours apart. Twelve hours after delivery of the second hormone, MII stage oocytes were isolated and injected with in vitro transcribed piggyBAC transposase mRNA (100 ng/ul) prepared in an injection buffer (5 mM Tris buffer, 0.1 mM EDTA, pH = 7.4). Decapitated sperm isolated from an 8 week old Gt(ROSA)26Sortm1.1(CAG=cas9*,EGFP)Fezh/J strain mouse (Jackson labs, PMID 25263330) was resuspended with the purified piggyBAC library in the same injection buffer at concentrations ranging from 0.5 to 1.4 ug/uL. Transposase-injected oocytes were then fertilized by piezo-actuated intracytoplasmic sperm injection (ICSI) as previously described (PMID 17406589). Injected embryos were cultured in 25 uL EmbryoMax® KSOM drops (Millipore) covered in mineral oil (Irvine Scientific) in batches of 25-50 embryos. After 84 or 96 hours, successfully cavitated blastocysts were screened for uniform fluorescence of the target sequence cassette and transferred into one uterine horn of 6-10 week old pseudopregnant CD-1 strain female mice (Charles River). Uterine transfer results in an ~24 hour lag, so the day of transfer was scored as E2.5 and embryos were dissected from euthanized animals 6 or 7 days later at ~E8.5 or E9.5, depending on the experiment.
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| Growth protocol |
Mouse embryos were cultured in vitro in KSOM at 37 ºC in 5% CO2 from the zygote to the blastocyst stage and subsequently transferred into the uterine horns of pseudopregnant females until isolation 6 or 7 days later.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Embryos were dissociated to single cell suspensions by adding 100 uL of TrypLE (Invitrogen, #12605010) and pipetting the embryo or embryo pieces every 5 minutes for ~30 minutes until complete dissociation was visually confirmed. Trypsin was deactivated by adding 100 uL PBS+BSA is added to the droplet and moving cells into a 1.5 mL eppendorf tube, followed by several rounds of additional collection with 100 to 200 uL of PBS+BSA to a final volume of 1 mL. Single cells were lysed in droplets using the 10x Chromium instrument.
10X Genomics Chromium Prep v2; PCR enrichment of target site amplicon from 10x cDNA pool. Refer to Methods in manuscript for primers sequences and PCR conditions.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
CRISPR-based lineage traced embryo
processed data file: embryo1_BestTree8420.nwt.txt
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| Data processing |
10x cellranger v2.0.1
Normalization in Seurat ("LogNormalize")
Genome_build: mm10
Supplementary_files_format_and_content: normalized UMI counts (mtx), gene names (tsv), and cellIDs (tsv); allele table containing filtered lineage tracer calls.
Supplementary_files_format_and_content: *nwt.txt: Newick files represent the best reconstructed tree. embryo2_BestTreeGreedy.nwt.txt represents the final tree for the embryo2a and embryo2b samples merged.
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| Submission date |
Aug 06, 2018 |
| Last update date |
May 15, 2019 |
| Contact name |
Michelle M Chan |
| Organization name |
UCSF
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| Lab |
Weissman Lab
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| Street address |
1700 4th St.
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE117542 |
CRISPR-based molecular recording of mouse embryogenesis |
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| Relations |
| BioSample |
SAMN09768132 |
| SRA |
SRX4510353 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3319520_embryo1_alleleTable.txt.gz |
113.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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