GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM1628331

Query DataSets for GSM1628331

Status

Public on Apr 13, 2015

Title

zf1_p8_S71

Sample type

SRA

Source name

50% Epiboly stage

Organism

Danio rerio

Characteristics

group: experimental batch 1

Growth protocol

Fertilized eggs from TL/AB in-crosses were incubated in 1 mg/ml pronase (Protease from Streptomyces griseus, Sigma-Aldrich) in a glass dish for 4–5 minutes until the chorion began to blister. The embryos were submerged in ~200 ml of embryo medium in a glass beaker without allowing them to contact air or plastic (both of which will cause the embryos to burst). The medium was poured off and new medium was vigorously added (again without allowing embryos to contact air) twice, in order to mechanically remove the chorions. The embryos were cultured in petri dishes coated with 2% agarose (to avoid contact with plastic) either at 23°C or 28°C until they reached 50% epiboly (about 6 hours post-fertilization at 28°C). At 50% epiboly, single embryos were visually confirmed to be at the correct stage

Extracted molecule

total RNA

Extraction protocol

Two pairs of watchmaker forceps were used to dissect the blastula cap of the embryo away from the yolk. First, one pair of forceps was used to hold and rotate the cap, while the other was used to cut and pinch away the yolk that extended below the blastula cap. Then, the blastula cap was cut slightly up the side, which exposed the yolk that was inside of the blastula cap, which could then be gently peeled away. The blastula cap was transferred by pipette into a microfuge tube that contained 60 μl of DMEM/F12 media. The cells were dissociated by vigorously flicking the tube 10 times, and then pipetting the entire volume twice while visually confirming that dissociation had occurred. A timer was started at this time to track the amount of time the embryo had been dissociated. If cell clumps were still visible, the tube was flicked again. 180 μl of DMEM/F12 was added to dilute the cell mixture, then 120 μl of the diluted cell mixture was pipetted across the surface of a new agarose-coated dish filled with DMEM/F12 media. To collect cells, a P2 pipettor was used, while observing the cells under the dissecting scope, to collect 0.5 μl of media that contained a single cell. This was pipetted into 3 μl of TCL lysis buffer (Qiagen) in the lid of a PCR strip and mixed 3 times to ensure that lysis occurred. After collection of 8 cells, the entire strip of lids was snapped into a 96-well plate and kept on dry ice.
We used a modified strand-specific single-cell RNA-seq protocol based on the SMART template switching method, where the template- switch oligonucleotide included a stretch of 5 randomized nucleotides, thereby tagging each mRNA molecule with a random molecular tag (RMT) prior to PCR to mitigate amplification bias. Furthermore, we used a modified library preparation protocol that shares similarities with Soumillon et al. and is based on the Nextera Sample Preparation Kit. It selectively amplifies the 5’ transcript end, retains strand information, and is compatible with standard Illumina sequencing primers.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

To map reads, we slightly modified the Zv9 reference transcriptome, since our reads were expected to originate from the 5’ end of each mRNA molecule, which could cause problems if the reference gene models had an incorrect annotation for the transcription start site (TSS). To address this, we extended the TSS for all Zv9 genes by 100 bases upstream to allow for minor fluctuations. We then aligned read-pairs directly to this modified reference transcriptome, using Bowtie with the following parameters: -q --phred33-quals -n 2 -l 25 -I 1 -X 2000 -a -m 200. As expected, following mapping, our reads overwhelmingly originated from near the TSS.
To quantify gene expression, we leveraged the 5 bp Random Molecular Tags (RMTs) that were associated with each sequencing read pair. For each annotated gene in Zv9, we identified all read-pairs that mapped to the correct strand, and collected each of the 5 bp RMTs that were associated with these reads. We collapsed duplicate RMT sequences together, in order to calculate the number of distinct RMTs associated with each gene. We quantified gene expression for 1,152 single cell libraries, and identified a subset of 207 failed/low-quality libraries with poor transcriptome complexity (< 2,000 genes detected per cell). After excluding these (remaining dataset of 945 cells), on average, we identified 47,000 unique molecules per sequencing library, corresponding to the detection of 3,400 genes per single cell.
Supplementary_files_format_and_content: zdata.expMatrix.txt --Expression matrix for all samples

Submission date

Mar 09, 2015

Last update date

May 15, 2019

Contact name

Rahul Satija

E-mail(s)

rsatija@nygenome.org

Phone

6177022468

Organization name

New York Genome Center