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Sample GSM1229072 Query DataSets for GSM1229072
Status Public on Feb 19, 2014
Title h_LF_iPS_rep2
Sample type SRA
 
Source name fibroblast, lung
Organism Homo sapiens
Characteristics cell type: iPS
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
 
Description replicate
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
Street address 3720 15th Ave NE
City Seattle
State/province WA
ZIP/Postal code 98195-5065
Country USA
 
Platform ID GPL10999
Series (1)
GSE50781 Primate iPS cells as tools for evolutionary analyses
Relations
BioSample SAMN02353891
SRA SRX348509

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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