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Sample GSM3580955

Query DataSets for GSM3580955

Status

Public on May 31, 2021

Title

Four-cell replicate 1 nuclear transfer

Sample type

SRA

Source name

nuclear transfer embryo

Organism

Bos taurus

Characteristics

embryo type: NT embryo
developmental stage: Four-cell

Extracted molecule

total RNA

Extraction protocol

Pools of 10 oocytes or embryos were chosen after visual inspection for each biological replicate to minimize variance caused by a single specimen. IVF and SCNT embryos were cultured in vitro under identical conditions (protocol IVF1; (Hiendleder S, et al. (2006) Tissue-specific effects of in vitro fertilization procedures on genomic cytosine methylation levels in overgrown and normal sized bovine fetuses. Biology of reproduction 75(1):17-23.)) to the blastocyst stage (day 7). The following cell stages were collected: GV and MII oocytes (one set used as reference for both IVF and SCNT embryos) and embryos at the four-cell, eight-cell, 16-cell, and blastocyst stage (both embryo types). Pools of 10 oocytes or embryos per stage were frozen and stored at ‑80 °C until analysis.
Frozen pools of 10 oocytes or embryos were thawed and lysed in 10 µL of lysis buffer (Prelude kit, NuGEN). The Ovation RNAseq v2 kit (NuGEN) was used for cDNA generation and single primer isothermal amplification (SPIA). The amplified cDNA was bead-purified (AmpureXP, Beckman-Coulter) and fragmented by sonication (Bioruptor, Diagenode; 25 cycles 30 seconds on/30 seconds off), and Illumina-compatible sequencing libraries were constructed from 500 ng of fragmented cDNA by ligation of adapters with sample-specific barcodes. The libraries were amplified (KAPA hifi polymerase, eight cycles, 95 °C 80 seconds, 55 °C 30 seconds, 72 °C 60 seconds) and quantified on a Bioanalyzer 2100 (Agilent) before being pooled at 10 nM concentration for multiplexed sequencing. Three biological replicates of each stage were sequenced on an Illumina GAIIx (single-read, mean coverage of 20 × 10e6 reads, 80-base read length).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer IIx

Description

non ribosomal RNA

Data processing

Reads were converted to Fastq using the CASAVA (v.1.6) base caller from Illumina.
Illumina adapters where clipped from each read using a custom script.
The first five bases were removed with a custom script from each read due to random priming effects
Poly-A tails were removed from the 3’-/5’-end of each read with a seed length of 10 nt. Reads with length <25nt after clipping were filtered out.
Reads were filtered from the 3′ and 5′ end with a quality cutoff of 20, and reads shorter than 30 were discarded
Reads were mapped against the reference genome (BosTau7) using Tophat2 (v.2.0.10) with default settings and the gene annotation obtained from Illumina’s iGenomes Project (UCSC: bosTau7).
Transcript abundance was counted using htseq-count (v. 0.6.1) with no strand specificity (-s no), union mode (-m union) and GFF attribute ‘gene’ (-i gene). The used gene annotation was obtained from Illumina’s iGenome Project (UCSC: bosTau7).
Genome_build: BosTau7
Supplementary_files_format_and_content: Tab-delimited text file containing the raw read counts per gene from HTSeq-count

Submission date

Jan 28, 2019

Last update date

May 31, 2021

Contact name

Alexander Graf

E-mail(s)

graf@genzentrum.lmu.de

Organization name

Ludwig-Maximilians-Universität München

Department

Gene Center Munich