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Sample GSM1099157

Query DataSets for GSM1099157

Status

Public on Sep 23, 2013

Title

A0571 embryo 4 extra-embryonic membranes

Sample type

SRA

Source name

embryonic day 13 extra-embryonic membranes

Organism

Monodelphis domestica

Characteristics

strain: LL2 mother (A0571) x LL1 father (A0578)
developmental stage: embryonic day 13
tissue: extra-embryonic membranes
Sex: female

Extracted molecule

total RNA

Extraction protocol

At 13 days post-copulation (d.p.c.), female opossums were anesthetized, the fetuses and respective extra-embryonic membranes (EEM) were collected and placed in either RNALater or PBS. Head/brain and EEM tissues were dissected for RNA extractions. After collection, the tissue was homogenized in TriReagent (Invirtogen) and total RNA was extracted using BCP (1-bromo-2 chloropropane), precipitated with isopropanol, and resuspended in RNase-free water. Potential gDNA contamination was removed by both DNase I treatment and the gDNA-removing column in Qiagen RNeasy Plus Mini kit (Qiagen, CA). RNA concentrations and A260nm/A280nm ratios were checked with a NanoDrop ND-1000 Spectrophotometer. The RNA quality was checked on both 1% agarose gel and Agilent 2100 Bioanalyzer.
The mRNA-seq libraries were made for brain and extra-embryonic membrane RNA samples with 1-3 µg total RNA input for four individuals in reciprocal F1 crosses and four individuals in the parental crosses, using Illumina TruSeq RNA Sample Prep Kit (Illumina Inc., CA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Illumina RNA-seq

Data processing

Illumina CASAVA 1.7 software was used for basecalling.
Adapter sequences were removed from the raw reads by custom scripts.
The short reads were aligned to the opossum reference genome (monDom5) using TopHat v1.4.1
The total expression level metric, FPKM (Fragments Per Kilobase-pair of exon Model), was calculated for all samples using Cufflinks v1.3.0 based on all mapped reads from the TopHat alignments. The multiple mapped reads were weighted using the “-u” parameter in Cufflinks. The expression level was normalized across the brain and extra-embryonic membrane samples using quartile normalization.
SNP positions were called in combined RNA-seq data from only uniquely mapped reads to the opossum reference genome with 40 of more coverage, using SAMtools software. Problematic SNPs with third allele, near indel positions or on the exon-intron junctions were removed. In total, we called 68k SNPs in the transcriptome. The reference and alternative allele counts were summarized at high quality X-linked SNP positions. We then filtered out high-coverage SNPs with 8 or more coverage in at least one of two female F1 individuals in both reciprocal crosses. To quantify the allele-specific expression in the brain and extra-embyronic tissues of the reciprocal crosses, at each identified SNP position we calculated the ratio of the number of reference allele containing reads over the total coverage.
Genome_build: UCSC monDom5
Supplementary_files_format_and_content: gzipped tab-delimited text file includes the FPKM value for expressed Ensembl v64 genes.

Submission date

Mar 15, 2013

Last update date

May 15, 2019

Contact name

Andrew Clark

E-mail(s)

[email protected]

Organization name

Cornell University

Street address

227 Biotech Building