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| Status |
Public on Jun 05, 2024 |
| Title |
DGM-00757_AM |
| Sample type |
SRA |
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| Source name |
alveolar macrophages
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| Organism |
Homo sapiens |
| Characteristics |
smoking status: nonsmoker
disease state: HIV-
cell type: alveolar macrophage
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from harvested nonsmoker, smoker and COPD alveolar macrophages was extracted with TRIzol with subsequent RNA clean-up using the RNeasy MinElute RNA purification kit to remove residual DNA
Using the reagents of the mRNA Sample Prep Kit and in accordance with the RNA sequencing protocol provided by Illumina, poly(A)+ mRNA was selected out from the total RNA samples using Sera-mag magnetic oligo(dT) beads. An RNA fragmentation kit (Ambion, Austin, TX) was used to fragment the mRNA, followed by first- and second-strand cDNA synthesis using random hexamer primers. An “end repair” reaction to blunt the ends of all fragments was then performed with Klenow polymerase and T4 DNA polymerase, and 3’- to 5’ exo-nuclease was used to create a 3’ adenine overhanging tail, facilitating the ligation of the amplification adapters. Ligation products were then separated on a 2% tris-acetate-EDTA-agarose gel for size selection, followed by purification with a gel extraction kit. The purified ligation products were then PCR amplified with complementary primers and the resultant cDNA was purified with QIAquick PCR kit (Qiagen), and the concentration was measured by the NanoDrop spectrophotometer.
Samples were then loaded onto Illumina flow cells for single end, 43 nucleotide, sequencing reactions
Images acquired by the Illumina Genome Analyzer II were analyzed by Firecrest and bases called by Bustard (both part of Illumina RTA pipeline version 1.6).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
alveolar macrophages obtained from bronchoalveolar lavage
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| Data processing |
Firecrest from Illumina RTA pipeline version 1.6 analyzed images acquired by the Illumina Genome Analyzer II.
Expression analysis was performed using Bowtie (v0.12, http://bowtie-bio.sourceforge.net)
Bowtie default parameters used were those in which the first seed alignment of size > 28 nt, allowing up to 2 mismatches, is chosen at random, and is used if it yields an alignment quality sum of > 70 with a maximum of 125 backtraces.
Resultant reads were aligned (mapped) to the reference genome build UCSC hg19 using Bowtie v 0.12
Bam files of aligned reads from Bowtie were imported into Partek Genomics Suite version 6.5
Aligned reads were mapped to RefSeq gene definition using Partek Genomic Suite.
To correct for transcript length and depth of coverage sequenced reads were converted into reads per kilobase of exon per million fragments sequenced (RPKM).
Genome_build: UCSC hg19
Supplementary_files_format_and_content: gene level rpkm data is a text file exported from Partek Genomics Suite that includes the RPKM values for 8 AM samples.
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| Submission date |
Dec 31, 2014 |
| Last update date |
Jun 05, 2024 |
| Contact name |
Yael Strulovici-Barel |
| E-mail(s) |
[email protected]
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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 |
GPL9115 |
| Series (2) |
| GSE64598 |
The Role of Interleukin-23 in the Early Development of Emphysema in HIV1+ Smokers [RNA-seq] |
| GSE64599 |
The Role of Interleukin-23 in the Early Development of Emphysema in HIV1+ Smokers |
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| Relations |
| BioSample |
SAMN03274203 |
| SRA |
SRX825957 |
| 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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