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Sample GSM3025412

Query DataSets for GSM3025412

Status

Public on Jan 01, 2019

Title

5dpa_4N_2

Sample type

SRA

Source name

5 dpa 4N

Organism

Ambystoma mexicanum

Characteristics

tissue: Axolotl limb (regenerating)
cellular fraction: 4N
time: 5 days post-amputation (dpa)

Extracted molecule

total RNA

Extraction protocol

Intact or regenerating limb tissue was microdissected into stump or epithelium fractions. Each fraction was chemically dissociated with either Trypsin-EDTA (epidermis) or collagenase/dispase (stump tissues). Each fraction was then stained with 10 ug/ml of DAPI. Stump tissues were then FACS sorted for 2N and 4N fractions. RNA was then isolated from all three fractions (stump 2N, stump 4N, and epidermis) using the Recoverall Total Nucleic Acid Isolation Kit for FFPE (Invitrogen). RNA quality was assessed on the Bioanalyzer 2100.
cDNA was generated using 10 ng of RNA input for the NuGEN Ovation RNA-seq System V2 protocol (Integrated Sciences). The quality and concentration of cDNA preps was then assessed using the Agilent DNA 1000 kit on the Bioanalyzer 2100 (Agilent Technologies). To prepare cDNA sequencing libraries, cDNA was first sheared to a peak size of 200 bp using the Covaris S220 (Covaris, Woburn, MA) according to the manufacturer’s protocol. Good quality and correct size distribution of sheared DNA fragments was assessed with the Agilent DNA High Sensitivity kit on the Bioanalyzer 2100 (Agilent Technologies). Sequencing libraries were then generated using the Wafergen PrepX Complete ILMN DNA Library kit (Takara Bio) protocol on the Apollo 324 NGS Library Prep System (Takara Bio). Quality of the DNA sequencing libraries was performed with the Agilent DNA High Sensitivity kit on the Bioanalyzer 2100 (Agilent Technologies).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

kallisto_counts_matrix.TMM.EXPR.matrix.tab
Two instrument models: Illumina Hiseq 2500 and Nextseq 500

Data processing

Samples were sequenced on one run of the Ilumina Nextseq 500 (300 cycle) and across 6 lanes of the Illumina Hiseq 2500 (raw read files for Hiseq were concatenated for ease of uploading).
The reads were aligned to a previously generated annotated axolotl de novo transcriptome (Bryant et al., 2017, GEO accession GSE92429). Alignment of samples and normalized TPMs were generated using Kallisto software (https://pachterlab.github.io/kallisto/) according to default parameters.
Differential expression analysis was performed using DESeq2 (https://bioconductor.org/packages/release/bioc/html/DESeq2.html).
Genome_build: GFBM000000000.1
Supplementary_files_format_and_content: tab delimited text file containing the normalized counts (TMM-normalized TPMs) for all samples (27 columns) for each transcript in the transcriptome (rows)

Submission date

Feb 27, 2018

Last update date

Jan 01, 2019

Contact name

Douglas A. Melton

E-mail(s)

dmelton@harvard.edu

Organization name

Harvard University

Street address

7 Divinity Ave.