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| Status |
Public on May 13, 2019 |
| Title |
Young_12_RNA |
| Sample type |
SRA |
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| Source name |
Bone marrow
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| Organism |
Homo sapiens |
| Characteristics |
cell type: HSCe (CD34+, CD38-,Lineage-)
donor age: 25
donor sex: M
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| Treatment protocol |
Miltenyi MACS magnetic bead purification was used to enrich frozen bone marrow MNC for CD34+ cells. The CD34+ fraction was then stained with CD2-TC, CD3-TC, CD4-TC, CD7-TC, CD8-TC, CD10-TC, CD11b-TC, CD14-TC, CD19-TC, CD20-TC, CD56-TC, and Glycophorin A-TC, followed by CD34-APC, CD38-PE-Cy7, CD123-PE, CD45RA-FITC and DAPI. Using a BD FACSAria I, HSCe (DAPI-, Lineage-, CD34+, CD38-) were FACS sorted into RLT+ from the Qiagen Allprep Micro Kit (#80284).
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| Extracted molecule |
total RNA |
| Extraction protocol |
HSCe RNA was extracted following the manufactures’ protocol for the Qiagen Allprep Micro Kit (#80284). Samples were eluted in 14 µL RNAse free water. An ethanol precipitation was then used to concentrate samples.
Samples with a RIN>8.5 were used for library preparation. Ribosomal RNA was removed using RiboGone (Clontech, #634846). Stranded libraries were prepared by the University of Michigan Sequencing Core using the SMARTer Stranded RNA-seq Kit (Clontech, #634836). Libraries were sequenced on the HiSeq-2500 with 50 bp pair-end sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Using Cutadapt (version 1.12), all reads were trimmed to 48 basepairs and adapters were removed.
Reads were aligned to the hg19 gencode v19 reference genome using the STAR aligner (version 2.5.2b), specifying the following parameters: outFilterType= BySJout, outFilterMultimapNmax=20, alignSJoverhangMin=8, alignSJDBoverhangMin=1 , outFilterMismatchNmax=999, alignIntronMin=20, alignIntronMax=1000000, alignMatesGapMax=1000000, alignEndsType=EndToEnd12.
Gene counts were calculated using QoRTs. QoRTs was run in secondstranded mode using the hg19 gencode annotation file without entries for ribosomal RNA.
Regularized log (rlog) normalized counts were generated using DESeq2. A multifactor design was used in order to control for sex of the donor as well as any batch effect during library preparation.
Genome_build: hg19
Supplementary_files_format_and_content: The HSCe_Normalized_Counts.txt file contains the regularized log-counts matrix generated by the DESeq2 rlog function. Prior to rlog transformation, a ddsHTSeq object was created with DESeq2 using a multifactor design controlling for donor sex and batch of library preparation. For each sample, a text file is also included that contains the raw counts generated by QoRTs.
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| Submission date |
Sep 28, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Maria E. Figueroa |
| E-mail(s) |
[email protected]
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| Phone |
305-243-7333
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| Organization name |
University of Miami Miller School Of Medicine
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| Department |
Human Genetics
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| Street address |
1501 NW 10th Ave, BRB 742F
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| City |
Miami |
| State/province |
Florida |
| ZIP/Postal code |
33136 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE104406 |
Aging Human Hematopoietic Stem Cells Manifest Profound Epigenetic Reprogramming of Enhancers That May Predispose to Leukemia (RNA-Seq of HSCe) |
| GSE104408 |
Aging Human Hematopoietic Stem Cells Manifest Profound Epigenetic Reprogramming of Enhancers That May Predispose to Leukemia |
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| Relations |
| BioSample |
SAMN07716875 |
| SRA |
SRX3229247 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2797288_Sample_56678.txt.gz |
214.1 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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