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Sample GSM1229077

Query DataSets for GSM1229077

Status

Public on Feb 19, 2014

Title

m_MF12_ES

Sample type

SRA

Source name

stem cells

Organism

Macaca fascicularis

Characteristics

cell type: ES

Growth protocol

All ESC and iPSC lines were cultured and expanded under standard ES-cell culture conditions in ko-DMEM supplemented with 20% knockout serum replacement, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% nonessential amino acid stock, and 15 ng/ml (cyiPSCs, goiPSCs, boiPS, MF12, R366.4), 10ng/ml (human iPSCs) and 50 ng/ml (human ESCs) basic fibroblast growth factor (all from Invitrogen, Carlsbad, USA)

Extracted molecule

polyA RNA

Extraction protocol

Cells were grown as described above in 6-well plates, removed from mouse feeder cells as good as possible and cells from one plate suspended in 1ml Trizol (Invitrogen). Total RNA from 500µl Trizol was isolated and further purified using RNAeasy columns (Quiagen).
RNA-Seq libraries were constructed using a custom protocol combining the Illumina TruSeq RNA protocol to generate cDNA and a double-indexing protocol [PMID: 22021376, PMID: 20516186] to generate sequencing libraries from this cDNA (full detailed protocol available upon request). Briefly, 1µg of total RNA was mRNA purified twice (Sera-Mag Magnetic Oligo(dT) beads, Thermo Scientific), cleaned up using RNAClean beads (Beckman Coulter), eluted in 2µl first strand buffer and 0.5µl random primers (Invitrogen) and fragmented for 7 min at 85°C. After first and second strand synthesis, cDNA was end-repaired, custom P5 and P7 adapters were ligated, blunted, quantified and amplified in an indexing PCR essentially as described [PMID: 20516186], except that both ends were indexed [PMID: 22021376]. The quality and concentration of the library were assessed on a BioAnalyzer.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer IIx

Data processing

Sequencing data was analyzed starting from BCL and CIF intensity files generated by the Illumina Genome Analyzer SCS 2.9/RTA 1.9 software. Ibis 1.1.6 was trained from the φX174 control reads of all lanes aligned to their reference sequence and then used to re-call bases and PHRED-like quality scores from intensity files [PMID: 19682367].
Only reads matching one of the used indexes or one-edit variants [PMID: 20516186] were further processed and subjected to an adapter and chimera filter; removing artifactual sequences and trimming adapter sequence starting at the read end [PMID: 21801405]. From this data, reads with more than 5 bases below a quality score of 15 as well as reads shorter than 60 bases after trimming were discarded.
Human Ensemblv66 gene models for GRCh37 were transferred to the coordinates of the chimpanzee genome (CHIMP2.1.4), the gorilla genome (gorGor3.1) and the rhesus macaque genome (MMUL_1) using Ensembl Compara v66 six primate EPO whole genome alignments. Since this version of Ensembl did not include a chimpanzee mitochondrial genome, the Ensembl Compara mitochondrial alignments were obtained from the v64 release. Further, the CHIMP2.1.4 genome was supplemented by the previously used mitochondrial genome. Exon start and end coordinates where independently transferred using the EPO alignments and only genes were used where (1) all exons and their respective start and end coordinates were assigned to a single chromosome/contig, (2) exons were on the same strand in each single species and (3) adjacent exons were not more than 1 Mb apart in the three species under consideration.
Tophat 1.4.1 [PMID: 19289445] was used to align sequences for each sample first to the inferred transcriptome of each species and the remaining reads to the reference sequence. We also aligned all samples to the mouse annotation (Ensembl v66) and mouse reference genome (musMus37). We removed all reads from the species alignments which aligned with the same or fewer mismatches to the mouse genome.
Gene models were quantified using Cufflinks 1.3.0 [PMID: 21697122] with activated fragment bias correction and correction for reads mapping to multiple genomic locations. For analysis, we only considered genes as detected with an FPKM value greater or equal to the size of the 95% FPKM-confidence interval [PMID: 22068331].
Genome_build: GRCh37, CHIMP2.1.4, gorGor3.1, MMUL_1
Supplementary_files_format_and_content: FPKM values for all ES/iPS samples

Submission date

Sep 11, 2013

Last update date

May 15, 2019

Contact name

Jay Shendure

Organization name

University of Washington

Department

Genome Sciences

Lab

Shendure