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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 07, 2020 |
| Title |
mouse_embryo_e6.75_handpickedcells_2_9 |
| Sample type |
SRA |
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| Source name |
Mouse embryo
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6 WT
tissue: Whole embryo
age: Embryonic day 6.75
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| Treatment protocol |
NA
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| Growth protocol |
Mice were kept in the animal facility of Institute of Experimental and Clinical Pharmacology and Toxicology, Faculty of Medicine, University of Freiburg, Freiburg, Germany
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| Extracted molecule |
total RNA |
| Extraction protocol |
Embryos were dissected in pre-warmed dissection medium (10% fetal calf serum (FCS) in DMEM/F12 containing Glutamax) and washed in pre-warmed PBS. For the E6.75 time point the extraembryonic part was cut off and a picture of each embryo was taken and single embryos was transferred into the wells of a pre-warmed non-adhesive 96-well plate containing 40 μl of TrypLE Express (Gibco 12604013). The wells were coated with FCS before adding the TrypLE. Embryos were incubated at 37°C for 10 minutes with pipetting up and down once in between and at the end to make a single cell solution. Dissociation was stopped with 120 μl of dissection medium and cells were centrifuged for 2 min at 1000 rpm in the 96-well plate. The supernatant was removed and cells from one embryo were resuspended in 200 μl cold PBS. For hand-picking, the drop containing the cells was placed in a plastic petri dish. Cells were picked under a Leica M165 FC binocular using ES-blastocyst injection pipettes (BioMedical Instruments, blunt, bent ID 15 μm, BA=35°) and placed into 1.2 μl lysis buffer containing polyT primer with unique cell barcode. Embryos from the E7.5 time point were cut under to chorion to include the extraembryonic mesoderm in the analysis. The embryos were imaged and the embryos of one or two litters were pooled and processed in a FCS coated eppendorf tube in the same way as the E6.75 embryos. After centrifugation the cells were resuspended in 200 μl PBS and kept on ice until flow sorting.
As described in CEL-Seq2 and mCEL-Seq2 protocol (Hashimshony et al. 2016 and Herman et al. 2018)
Adapted from TruSeq Small RNA Library Preparation Protocol
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
library of 96 cells
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| Data processing |
For image aquisition, intensity extraction and basecalling HiSeq Control Software 2.0.2, RTA 2.4.11 / Recipe Fragment 2.0.0.2 was used. Conversion of bcl2fastq files was performed using bcl2fastq 2.17.1.14
Paired end reads were aligned to the transcriptome using bwa (version 0.6.2-r126) with default parameters. The transcriptome contained all gene models based on the mouse ENCODE VM9 release downloaded from the UCSC genome browser comprising 57,207 isoforms derived from 57,207 gene loci with 57,114 isoforms mapping to fully annotated chromosomes (1 to 19, X, Y, M). All isoforms of the same gene were merged to a single gene locus. Furthermore, gene loci overlapping by >75% were merged to larger gene groups. This procedure resulted in 34,111 gene groups.
The right mate of each read pair was mapped to the ensemble of all gene loci and to the set of 92 ERCC spike-ins in sense direction. Reads mapping to multiple loci were discarded. The left read contains the barcode information: the first six bases correspond to the cell specific barcode followed by six bases representing the unique molecular identifier (UMI). The remainder of the left read contains a poly(T) stretch. The left read was not used for quantification. For each cell barcode, the number of UMIs per transcript was counted and aggregated across all transcripts derived from the same gene locus.
Based on binomial statistics, the number of observed UMIs was converted into transcript counts (Gruen et al., 2014).
Genome_build: ENCODE VM9
Supplementary_files_format_and_content: CSV files, columns represent each cell barcode (total barcodes used = 192), rows represent the geneid and the values in the file are the quantified number of transcripts.
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| Submission date |
Jun 04, 2020 |
| Last update date |
Dec 08, 2020 |
| Contact name |
Sagar - |
| E-mail(s) |
sagar@uniklinik-freiburg.de
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| Organization name |
University Medical Center Freiburg
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| Department |
Department of Internal Medicine II
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| Lab |
Sagar
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| Street address |
Hugstetter Straße 55
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| City |
Freiburg |
| ZIP/Postal code |
79106 |
| Country |
Germany |
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| Platform ID |
GPL21493 |
| Series (1) |
| GSE151824 |
Spatiotemporal sequence of mesoderm and endoderm lineage segregation during mouse gastrulation |
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| Relations |
| BioSample |
SAMN15102451 |
| SRA |
SRX8472447 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4591482_mouse_embryo_e6.75_handpickedcells_2_9.coutt.csv.gz |
919.7 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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