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| Status |
Public on May 08, 2014 |
| Title |
DGM-10568_LA-BC |
| Sample type |
SRA |
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| Source name |
airway basal cells, nonsmoker
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| Organism |
Homo sapiens |
| Characteristics |
cell type: airway basal cells
smoking status: nonsmoker
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| Growth protocol |
Airway epithelial cells collected by brushing were cultured supplemented with growth factors according to the manufacturer's instructions. Media was changed every 2 days until time of harvest at day 7, when the cells had reached 70 to 80% confluence.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from harvested nonsmoker and smoker basal cells was extracted with TRIzol with subsequent RNA clean-up using the RNeasy MinElute RNA purification kit to remove residual DNA.
The TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA) was utilized as per protocol to generate cDNA that was then purified with QIAquick PCR purification kit (Qiagen, Valencia, CA).
Samples were loaded onto an Illumina flowcell for paired-end 101 x 2 sequencing reactions using the Illumina HiSeq 2000, using a single flow cell with 4 samples multiplexed per lane of flow cell.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
DGM-10568
Basal cells cultured from airway epithelium samples.
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| Data processing |
HiSeq Control Software (Illumina) was used to perform image analysis, base calling and sequence analysis on HiSeq generated sequence images resulting in fastq files.
Expression analysis was performed using Bowtie (v0.12.8.0, http://bowtie-bio.sourceforge.net), Tophat (v2.0.4, http://tophat.cbcb.umd.edu) and Cufflinks (v2.0.2, http://cufflinks.cbcb.umd.edu).
To correct for transcript length and depth of coverage, raw paired-end sequenced reads were converted into fragments per kilobase of exon per million fragments sequenced (FPKM) resulting in the fpkm_tracking files.
Resultant fragments were aligned (mapped) to the reference genome build UCSC hg19 using Bowtie.
Non-aligned reads were segmented using Tophat and re-aligned to the genome, aligning reads that span introns and determining junction splice sites. Cufflinks assembled reads into transcripts and assembled reads merged using Cuffmerge; the reads generated from a transcript are directly proportional to the transcripts relative abundance. For the analysis, Tophat (which automatically runs Bowtie) was run for each read pair in a given lane.
For each read pair, Tophat produced one file which contained all reads mapped to the human_g1k_v37 genome and RefSeq (http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/refGene.txt.gz) transcriptome.
This process was carried out independently for each sample lane. Before running Cufflinks, Samtools (v0.1.18.0, http://samtools.sourceforge.net) was used to merge each sample's independent Tophat results into one file. This file contains all of the aligned reads for each read pair. Finally, Cufflinks was run on the merged Tophat output to produce a file with FPKM values for each sample.
Data was analyzed using Tuxedo pipeline software.
Genome_build: GRCh37
Supplementary_files_format_and_content: fpkm_tracking files are text files that include the RefSeq gene and location, the FPKM value, high and low confidence and status.
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| Submission date |
Jun 06, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Yael Strulovici-Barel |
| E-mail(s) |
yas2003@med.cornell.edu
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| Organization name |
Weill Cornell Medical College
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| Department |
Department of Genetic Medicine
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| Lab |
Crystal
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| Street address |
1300 York Avenue
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10021 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE47718 |
Smoking Dysregulates the Human Airway Basal Cell Transcriptome at COPD-linked Risk Locus 19q13.2 |
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| Relations |
| Reanalyzed by |
GSM2276644 |
| BioSample |
SAMN02191973 |
| SRA |
SRX297206 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1155376_DGM-10568_LA-BC_genes.fpkm_tracking.gz |
696.6 Kb |
(ftp)(http) |
FPKM_TRACKING |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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