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Sample GSM1911769

Query DataSets for GSM1911769

Status

Public on Nov 26, 2016

Title

34_ICAR_SCNT_61

Sample type

SRA

Source name

Endometrium_Inter-caruncle_SCNT_34d

Organism

Bos taurus

Characteristics

tissue source: Endometrium
tissue: Inter-caruncle
gestation day: 34
group: somatic cell nuclear transfer (SCNT)

Extracted molecule

polyA RNA

Extraction protocol

Total RNA was extracted from snap frozen tissues using the guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 2006) (TRIzol® Reagent, Invitrogen, Carlsbad, CA) following the manufacturer’s instructions with slight modifications. A purification with acid phenol:chloroform (pH 4.5, Ambion) was added prior to RNA precipitation with isopropanol. RNA samples were purified with the RNeasy® Mini Kit (Qiagen, Boston, MA), quantified, and assayed for purity on a 2100 Bioanalyzer (Agilent, Santa Clara, CA).
Three µg of total RNA was used for the polyA+ library construction using a TruSeq SBS Sequencing Kit Version 3

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

Base-calling was performed by Casava v1.8.2
Sequences were aligned to the cattle (Bos taurus) genome (Btau4) using the TopHat software, allowing for 2 mismatches for each block of 25 nucleotides. The unaligned sequences were trimmed at the 3’ end to 75 nt and realigned; the process was then repeated once more, with unaligned reads trimmed to 50 nt in length.
The three files with aligned sequences were merged, and the sequences were filtered to retain those matched to a unique position in the reference genome, with mapping quality greater than 30 and containing up to 3 mismatches.
Duplicate reads were removed by Picard tools
Sequence reads were counted according to overlaps on exon models obtained from the ensGene database from the UCSC genome browser. The database was downloaded in January 2011 using R software and processed using the Bioconductor packages rtracklayer, Rsamtools, GenomicRanges and GenomicFeatures. A set of genes that was not annotated in the Ensembl database but that was annotated in the refGene database from the UCSC genome browser was analyzed in conjunction with the Ensembl annotation. The reads that overlapped to an exon model were used for downstream analyses.
Genome_build: btau4
Supplementary_files_format_and_content: unfiltered_reads_count_EET.txt: reads count for extra-embryonic tissues and exon length
unfiltered_reads_count_END.txt: reads count for endometrial tissues and exon length

Submission date

Oct 19, 2015

Last update date

May 15, 2019

Contact name

Fernando H Biase

E-mail(s)

fbiase@vt.edu

Phone

540-231-9520

Organization name

Virginia Tech