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Sample GSM5282491

Query DataSets for GSM5282491

Status

Public on Aug 18, 2021

Title

scRNA-seq of Wild type embryo replicate 1

Sample type

SRA

Source name

tail

Organism

Danio rerio

Characteristics

treatment: WT

Treatment protocol

Embryos were incubated until the 10-12 somite stage and then dissected in ice cold Hank’s Balanced Salt Solution. The tail was collected by cutting immediately posterior to the last formed somite. Groups of tails consisting of ten tails for wild-type, FGF, or BMP inhibition or twelve tails for Wnt inhibition were pooled together.

Extracted molecule

total RNA

Extraction protocol

Cells were dissociated by incubation in 20 U/mL papain solution (Worthing Biochemical) for 15 minutes at 29 °C with gentle agitation. Halfway through the incubation the solution was triturated ten times with a P200 pipette. Cells were spun down at 300g for five minutes and then resuspended in 40 L of cold HBSS. Cell concentration and viability were checked with a hemocytometer and the volume of the solution was adjusted if required.
Single cell suspension in RT Master Mix was loaded on the Single Cell A Chip and partition with a pool of about 750,000 barcoded gel beads to form nanoliter-scale Gel Beads-In-Emulsions (GEMs). Each gel bead has primers containing (i) an Illumina R1 sequence (read 1 sequencing primer), (ii) a 16 nt 10x Barcode, (iii) a 10 nt Unique Molecular Identifier (UMI), and (iv) a poly-dT primer sequence. Upon dissolution of the Gel Beads in a GEM, the primers are released and mixed with cell lysate and Master Mix. Incubation of the GEMs then produces barcoded, full-length cDNA from poly-adenylated mRNA. Silane magnetic beads were used to remove leftover biochemical reagents and primers from the post GEM reaction mixture. Full-length, barcoded cDNA was then amplified by PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon size prior to library construction. R1 (read 1 primer sequence) were added to the molecules during GEM incubation. P5, P7, a sample index, and R2 (read 2 primer sequence) were added during library construction via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina bridge amplification. The Single Cell 3’ Protocol produces Illumina-ready sequencing libraries. A Single Cell 3’ Library comprises standard Illumina paired-end constructs which begin and end with P5 and P7. The Single Cell 3’ 16 bp 10x Barcode and 10 bp UMI are encoded in Read 1, while Read 2 is used to sequence the cDNA fragment. Sequencing a Single Cell 3’ Library produces a standard Illumina BCL data output folder. The BCL data includes the paired-end Read 1 (containing the 16 bp 10x Barcode and 10 bp UMI) and Read 2 and the sample index in the i7 index read.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

No other description

Data processing

We aligned the scRNA-seq data to Grcz11 and demultiplexed using Cell Ranger (10X Genomics, version 3.0).
Genome_build: Grcz11
Supplementary_files_format_and_content: Matrix market file for UMI counts. TSV file for annotation such as clustering results. TXT files for cell barcodes (column labels for MTX file) and gene names (row labels for MTX file).

Submission date

May 05, 2021

Last update date

Aug 11, 2023

Contact name

Scott A. Holley

E-mail(s)

kojima.yasuhiro@med.nagoya-u.ac.jp

Organization name

Yale University

Department

Department of Molecular, Cellular and Developmental Biology