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Sample GSM2722412

Query DataSets for GSM2722412

Status

Public on Aug 01, 2017

Title

endo3

Sample type

SRA

Source name

stage 12-14 Xenopus laevis embryos

Organism

Xenopus laevis

Characteristics

cell type: Endoderm

Extracted molecule

total RNA

Extraction protocol

Primordial germ cells and stage-matched endoderm cells were isolated from stage 12-14 Xenopus laevis embryos and collected into RNA lysis buffer as described by Butler et al. (2016). Total RNA was extracted using the RNAqueous-Micro Total RNA Isolation Kit per the manufacturer’s protocol (Ambion).
Three 1ng aliquots of each rRNA-depleted sample were used as template for preparation of sequence-able template DNA molecules using the ScriptSeq v2 RNASeq Library Preparation Kit (Illumina, Part # SSV21124). The quality and size distribution of the amplified libraries were determined utilizing an Agilent 2100 Bioanalyzer High Sensitivity DNA Chip. Libraries were quantified using the KAPA Library Quantification Kit (Kapa Biosystems, Boston, MA), and equimolar concentrations were diluted prior to loading onto the flow cell of the Illumina cBot cluster station. The libraries were extended and bridge amplified to create sequence clusters using the Illumina HiSeq PE Cluster Kit v4, and sequenced on an Illumina HiSeq Flow Cell v4 with 100-bp paired-end reads plus index read using the Illumina HiSeq SBS Kit v4. Real time image analysis and base calling were performed on the instrument using the HiSeq Sequencing Control Software version 2.2.58. Three DNA libraries were prepared from Xenopus laevis primordial germ cell RNA and three from matched endoderm cell RNA.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Raw reads from PGC and endoderm samples were aligned to each genome version using Tophat2 v2.1.0
Aligned reads were sorted and counted with Samtools v1.2 and HTSeq v0.6.0, respectively.
Raw count data were filtered to remove transcripts with FDR > 0.1, and CPM < 3 in at least three samples.
Filtered counts were TMM normalized and GLMTagwise dispersion was estimated.
Data from alignment to v7.1 and v9.1 were merged and normalized generating 13,469 total unique transcripts. Note: for transcripts identified in both genome versions, the counts from v9.1 were used.
EdgeR v3.12.0 was used for differential gene expression analysis. Transcripts were considered to be differentially expressed if the FDR corrected (Benjamini–Hochberg) p value was < 0.05.
Bowtie2 v2.2.6 was used to create index reference genomes for Xenopus laevis v7.1 and v9.1 (xenbase.org, September, 2015)
Genome_build: 7.1 and 9.1
Supplementary_files_format_and_content: csv

Submission date

Jul 31, 2017

Last update date

May 15, 2019

Contact name

Mary Lou King

E-mail(s)

MKing@med.miami.edu

Organization name

University of Miami, Miller School of Medicine

Department

Cell Biology