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| Status |
Public on Apr 18, 2018 |
| Title |
Knockout replicate 4 |
| Sample type |
SRA |
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| Source name |
embryonic ferret forebrain
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| Organism |
Mustela putorius furo |
| Characteristics |
dropseq batch: run3
aspm genotype: knockout
litter: 5
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| Extracted molecule |
total RNA |
| Extraction protocol |
embryos removed from anesthetized jill, forebrains dissected out in cold HBSS, minced and cryopreserved (gradually frozen in HBSS w/ 10%DMSO & stored at -80); shipped on dry ice then thawed and dissociated using trypsin to a single-cell suspension on the day of Droplet capture
standard Illumina paired-end library
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Drop-seq
see Macosko et al. 2015 for the Drop-seq library strategy. Briefly, paired end libraries include a sample (biological replicate) barcode and a unique molecular identifier on read 1 (R1) and a 50-base read from the 3'-end of the captured cDNA molecule on read 2 (R2).
As per dropseq protocol, R1 are barcodes and molecular identifiers, and R2 is the sequence, so paired reads do nor represent two reads with an insert
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| Data processing |
Tophat alignment version 2.1.1 of paired end raw bulk data
Transcriptome assembly with cufflinks version 2.2.1
The assembled transcriptome and the Ensembl reference transcriptome version 1.0.85 were merged using cuffmerge from cufflinks 2.2.1, this merged reference file was then used for the single cell RNA-seq analysis in the Drop-Seq pipeline.
Single Cell Fastq reads were converted to BAM using the “FastqToSam” command in Picard version 1.130
For the single cell raw data, the Drop-Seq Core Computational Protocol version 1.0.1 was followed to obtain the Digital Gene Expression Matrix. Read pairs where more than one base in the barcode had a quality below 10 were discarded. Adapter sequences were trimmed from the 5’ end of the read, along with polyA tails. Star-2.5.2 was used to map reads to the custom transcriptome reference. The digital gene expression matrix was extracted using the “DigitalExpression” program of the Drop-seq protocol, keeping only cells with at least 200 reads per cell for clustering analysis.
Genome_build: MusPutFur1.0, transcript annotation version MusPutFur1.0.85
Supplementary_files_format_and_content: The SAMPLE_out_gene_exon_tagged.dge.txt.gz files are the Digital Gene Expression Matrices obtained from the Drop-Seq Core Computational process and are ready to be normalized, filtered, and analyzed. We do not provide the DGE of the sample which was not included in the analysis due to it being an outlier when clustering, but we have included the raw single cell sequencing data for that sample.
Supplementary_files_format_and_content: The CufflinksID_coordinates.txt is a text file with the ferret genome coordinates of some of the genes that we detected in bulk sequencing experiment and added to the reference transcriptome of the ferret, since these genes are not annotated in the current ferret reference transcriptome. These can be used along with the Digital Gene Expression files for genes that do not have annotation to look up their position in the ferret genome. In some cases it is then possible to infer which gene it might be based on conservation with other species that have this gene annotated.
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| Submission date |
Feb 01, 2018 |
| Last update date |
May 16, 2018 |
| Contact name |
Byoung-Il Bae |
| Organization name |
Yale University
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| Street address |
Department of Neurosurgery, School of Medicine, Yale University
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| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06510 |
| Country |
USA |
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| Platform ID |
GPL24172 |
| Series (1) |
| GSE110010 |
Single-cell RNA-seq of ASPM mutant ferret embryonic forebrain |
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| Relations |
| BioSample |
SAMN08452128 |
| SRA |
SRX3639934 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2976351_KO_5G_out_gene_exon_tagged.dge.txt.gz |
13.7 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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