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| Status |
Public on Feb 13, 2014 |
| Title |
HL2_DGE-seq |
| Sample type |
SRA |
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| Source name |
Lymphoblastoid cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Lymphoblastoid
biopsy site: blood, peripheral vein
coriell catalog #: GM02184
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| Biomaterial provider |
http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=GM02184
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| Growth protocol |
We obtained two cell types from Coriell for this study: skin fibroblast cells and lymphoblastoid cell lines (LCLs). Primary skin fibroblast cells from three human, three chimpanzee, and three macaque individuals. LCLs, which are B cells immortalized with Epstein-Barr Virus, were obtained from the same three human and three chimpanzee individuals that fibroblasts were isolated from. EBV does not reliably transfect macaque lymphocyte cells, so matched macaque LCLs cells were not available for this study. Importantly, other recent genome-wide studies that used macaque LCLs were of B-Lymphocyte cells transformed with rhesus herpes papio virus, a close relative of human EBV. Cells from all species were grown in standard growth media. Fibroblast growth media consisted of Gibco's MEM (10370-021), L-Glutamine (25030-081), Pen/Strep (15140-122), and 10% FBS (Hyclone SH30070). LCLs growth media consisted of Gibco's RPMI (21870) media with L-Glutamine, Pen/Strep, and 15% FBS. We harvested ~35 million cells for DNase assays and 5 million for RNA DGE-seq expression analysis.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Standard Trizol extraction protocol followed by Qiagen's DNase treatment and column purification protocol.
Illumina's DGE library protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
nla_s_1_sequence.txt
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| Data processing |
library strategy: DGE-seq (Digital Gene Expression)
Illumina CASAVA 1.3 software used for single-end basecalling.
Sequenced reads were trimmed to the first 16bp to remove adaptor sequence using custom scripts, then 4 bp sequences accounting for the NlaIII cut site (CATG) were added to the 3'-end to construct a 20 mer final sequence length. Next, reads were masked for low-complexity or low-quality sequence, then mapped to the samples native whole genome (hg19 for human, panTro2 for chimpanzee and rheMac2 for macaque) using BWA version 0.6.1-r104 with default indexing (index -a bwtsw) and aln parameters (aln -t 8) to create alignment SAI files.
SAM files were created from the SAI files using bwa samse with default parameters, then filtered for alignments with greater than 4 mismatches using custom scripts.
The SAM files (after filtering for mismatches >4) were converted to BAM format using "samtools view", then sorted via chromosome using "samtools sort", then converted to BED format using "bedtools bamtobed".
Using custom scripts, we compared the DGE-seq sequence counts that aligned to exonic regions within each species' native sequence alignment.
Genome_build: hg19, panTro2, rheMac2
Supplementary_files_format_and_content: Tab delimited txt file, comprised of a matrix of 16 columns by 19233 rows. First column is the gene name, followed by 15 columns of sequence read counts that fell within an exon of each individual gene. There are 3 biological replicates of human fibroblast (hf), chimpanzee fibroblast (cf), macaque fibroblast (qf), human LCLs (hl), and chimpanzee LCLs (cl), ordered respectively from left to right.
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| Submission date |
Feb 12, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Yoichiro Shibata |
| E-mail(s) |
yoichiro.shibata@gmail.com
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| Organization name |
Duke University
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| Department |
Inst. of Genome Sciences and Policy
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| Lab |
Greg Crawford
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| Street address |
101 Science Dr
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| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27708 |
| Country |
USA |
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| Platform ID |
GPL10999 |
| Series (2) |
| GSE54906 |
Extensive Evolutionary Changes in Regulatory Element Activity during Human Origins Are Associated with Altered Gene Expression and Positive Selection [DGE-seq] |
| GSE54908 |
Extensive Evolutionary Changes in Regulatory Element Activity during Human Origins Are Associated with Altered Gene Expression and Positive Selection |
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| Relations |
| BioSample |
SAMN02640518 |
| SRA |
SRX469938 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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