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Sample GSM2113459

Query DataSets for GSM2113459

Status

Public on Sep 12, 2016

Title

Sexual Females rep3

Sample type

SRA

Source name

Sexual Females_whole carcass

Organism

Daphnia pulex

Characteristics

clone: PA42
developmental stage: Pre-ephipial females
age: 8 days (±1)
tissue: Whole carcass

Growth protocol

Females were maintained in 3L containers containing COMBO media diluted 1:1 with H2O at 20C. All females were fed Scenedesmus at approximately 100,000 cells/mL. New offspring were removed from the containers daily and transferred to new containers. Males, asexual females and sexual females were isolated from culture seperately using differentially-sized strainers and visual identification under a dissecting microscope.

Extracted molecule

total RNA

Extraction protocol

Collections of whole D. pulex individuals (~50-75) were homogenized using a small pestle in microcentrifuge tubes containing lysis buffer. Total RNA was isolated using solid phase extraction (Bioline, Inc.). Each sample was then snap-frozen in liquid nitrogen and stored at -80C.
Library construction utilized the nAnT-iCAGE protocol (Murata et al., 2014). Briefly, first-strand reverse transcription was performed with total RNA from each sample using a random primer (N6 plus 3). The 5' m7G cap was oxidized (with sodium peroxide) and biotinylated using biotin (long arm) hydrazide. Biotinylated RNA/cDNA hybrid molecules were pulled down using magnetic streptavadin beads. After RNase-treatment of captured hybrids, a barcoded linker oligo was ligated to the 5' end of the cDNA molecules, follwed by ligation of a 3' linker. Second-strand synthesis was completed using primers that anneal to the 5' linker and anchor primer, creating the final dsDNA product.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

CAGE

Instrument model

Illumina HiSeq 2000

Data processing

Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence and de-multiplexed using the FASTX Toolkit (fastx_barcode_splitter.pl; http://hannonlab.cshl.edu/fastx_toolkit/). Reads from each sample were iteratively mapped to the D. pulex Dappu1 nuclear genome assembly using bwa aln 0.6.2-r126 with parameters -t16 -B 3 -n 3 followed by bwa samse. Barcodes (3nt) were removed from reads prior to upload using the FASTX Toolkit (fastx_trimmer).
Ribosomal RNA reads (28S, 18S and 5S) were removed by rRNAdust (fantom.gsc.riken.jp/5/suppl/rRNAdust/).
CAGE-dectected TSSs (CTSSs) from each sample were normalized by fitting the data to a power-law distribution (Balwierz et al.,2009) using CAGEr (Haberle et al., 2015).
Abundance measurments of CTSSs in raw counts and tags per million (tpm) from each sample were made using CAGEr (Haberle et al., 2015).
Genome_build: Dappu1 (http://genome.jgi.doe.gov/Dappu1/Dappu1.home.html)
Supplementary_files_format_and_content: tab-delimited abundance files include the coordinates of CTSSs and their normalized abundance in tags per million (tpm)

Submission date

Apr 11, 2016

Last update date

May 15, 2019

Contact name

R. Taylor Raborn

E-mail(s)

rtraborn@asu.edu

Organization name

Arizona State University

Department

Center for Mechanisms of Evolution