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Sample GSM1064828 Query DataSets for GSM1064828
Status Public on Jan 19, 2014
Title hsa pl M1 [batch1]
Sample type SRA
Source name Placenta
Organism Homo sapiens
Characteristics Sex: male
strain: NA
age: embryo, 13 weeks post-conception
extract_protocol: Illumina GAIIx, strand-specific
library-type (for tophat and cufflinks): fr-secondstrand
Extracted molecule polyA RNA
Extraction protocol Total RNA was extracted using the Trizol (Invitrogen) procedure or RNAeasy/RNAeasy Lipid/miRNeasy (Qiagen) column purification kits. RNA quality was assessed using an Agilent 2100 Bioanalyzer.
Non-strand-specific sequencing libraries were prepared using the mRNA-Seq Sample Prep Kit (Illumina) according to the manufacturer's instructions. Briefly, polyadenylated RNA was isolated using a poly-dT bead procedure and then chemically fragmented and randomly primed for reverse transcription. After second-strand synthesis, the ends of the double-stranded cDNA were repaired. After 3'-end adenylation of these products, Illumina Paired-End Sequencing adapters were ligated to the blunt ends of the cDNA fragments. Ligated products were run on gels; 250-300 bp fragments were excised and then PCR-amplified (15 cycles). After column-purification, qualities of the resulting libraries were assessed using Agilent 2100 Bioanalyzers. Strand-specific libraries were prepared using an optimized version of the original Directional mRNA-seq Library Prep Pre-Release Protocol from Illumina. We used reagents from various companies instead of Illumina’s to decrease significantly the library cost and as a consequence adapted the reaction parameters for each step. In addition, we integrated a size selection step, right after fragmentation to optimize the insert size distribution and decrease the adapter contamination. This optimized protocol is available upon request.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina Genome Analyzer IIx
Data processing The basecalling was performed with the Illumina GAIIx pipeline, version 1.8 and with Ibis (Kircher et al, 2009). Ibis was used on non-strand specific samples and on hsa br M 2ss.. The quality values are given in the phred-33 format, for all samples.
Raw reads were aligned with TopHat version 1.4.0 and Bowtie version 0.12.5. Command line for TopHat: tophat -p 4 -o -a 8 -i 40 -m 1 -I 1000000 -F 0 --coverage-search --microexon-search.
Splice junctions were detected from TopHat unambiguous read alignments, requiring an anchor size of at least 8bp and at most 1 mismatch per alignment.
The genome-wide read coverage was determined using the "wiggles" utility in TopHat.
Expression levels for Ensembl-annotated genes were estimated with Cufflinks 2.0.0, using all reads, with the multi-read correction embedded in Cufflinks.Cufflinks command line: cufflinks -q -G Transcripts_ProteinCoding.gtf -p 4 --library-type ${librarytype} --multi-read-correct. For strand-specific samples, the library-type was set at fr-secondstrand (batch 1) or fr-firststrand (batch 2), while for non-strand-specific samples the library-type was set at fr-unstranded. For expression level computation, GTF files were retrieved from Ensembl 62, and filtered to retain only transcripts annotated as "protein-coding" for protein-coding genes (all transcripts were kept for other gene biotypes).
Genome_build: hg19, gorGor3, MMUL_1, mm9, monDom5, ornAna1, galGal3, JGI 4.2
Supplementary_files_format_and_content: .FPKM files contain expression levels quantified with Cufflinks; the columns are tab-separated. .bedGraph files contain genome-wide read coverage statistics, in bedGraph format, obtained with the "wiggles" utility in TopHat.
Submission date Jan 15, 2013
Last update date May 15, 2019
Contact name Anamaria Necsulea
Organization name CNRS, Université Claude Bernard Lyon 1
Department Laboratoire de Biométrie et Biologie Evolutive
Street address 43 Bd. du 11 Novembre 1918
City Lyon
ZIP/Postal code 69622
Country France
Platform ID GPL10999
Series (1)
GSE43520 The evolution of lncRNA repertoires and expression patterns in tetrapods
SRA SRX217683
BioSample SAMN01886754

Supplementary file Size Download File type/resource
GSM1064828_hsa_pl_m1.FPKM.txt.gz 1.2 Mb (ftp)(http) TXT
GSM1064828_hsa_pl_m1.bedGraph.gz 89.8 Mb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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