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Sample GSM2976359

Query DataSets for GSM2976359

Status

Public on Apr 18, 2018

Title

Bulk progenitors replicate 2

Sample type

SRA

Source name

newborn ferret forebrain

Organism

Mustela putorius furo

Characteristics

dropseq batch: not applicable
aspm genotype: wildtype
litter: 2

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

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

bulk RNA-seq used only to generate reference transcriptome
CufflinksID_coordinates.txt
Illumina TrueSeq v2

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.

Submission date

Feb 01, 2018

Last update date

May 16, 2018

Contact name

Byoung-Il Bae

Organization name

Yale University

Street address

Department of Neurosurgery, School of Medicine, Yale University

City

New Haven