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| Status |
Public on May 13, 2019 |
| Title |
AR010 |
| Sample type |
SRA |
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| Source name |
PB CD34+ cells_sgCTRL
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| Organism |
Homo sapiens |
| Characteristics |
donorid: 3565
age: 35
Sex: F
tissue: Peripheral blood (PB)
cell type: CD34+
treatment: sgCTRL
sample: E10
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| Treatment protocol |
Cells were transfected with equimolar amounts of Cas9 protein (PNA Bio, 1 μg/μL in PBS) and sgRNAs using the Neon Transfection System (Thermo Fisher). Electroporation conditions used were 1600 volts, 10 milliseconds, and 3 pulses. For controls, CD34+ cells were transfected with equimolar amounts of Cas9 protein (PNA Bio, 1 μg/μL in PBS) and a non-targeting sgRNA. Following transfection, cells were cultured in cytokine supplemented medium (IMDM + 20% Human Serum AB + hTPO (100 ng/ml), hSCF (100 ng/ml), hIL-6 (20 ng/ml), hFLT-3 (10 ng/ml) and non-essential amino acids for 72 hrs under low oxygen conditions. CD34+ cells were then FACS isolated and cultured using the above media and low oxygen conditions. After 72 hrs in culture, cells were used for RNA extraction.
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| Growth protocol |
Frozen PB CD34+ cells (n=4 replicates) were thawed and cultured in IMDM + 20% Human Serum AB + hTPO (100 ng/ml), hSCF (100 ng/ml), hIL-6 (20 ng/ml), hFLT-3 (10 ng/ml) and non-essential amino acids for 72 hrs under low oxygen conditions.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the Qiagen Allprep Micro kit (Qiagen, #80204), following the manufacturer’s instructions.
Stranded ribodepelted libraries were prepared by the University of Miami Sequencing Core using the Illuminia TruSeq Stranded Total RNA kit (Illumina, #20020596). ERCC spike-ins were included for library preparation. Libraries were sequenced on the HiSeq-3000 with 75 bp paired-end sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
Using Cutadapt (version 1.12), all reads were trimmed to 72 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=EndToEnd.
Gene counts were calculated using QoRTs. QoRTs was run in stranded mode using the hg19 gencode annotation file that included ERCC spike-in information, but did not include entries for ribosomal RNA, mitochondrial RNA, or tRNA.
Genome_build: hg19
Supplementary_files_format_and_content: The processed file is a text file that contains the raw counts generated by QoRTs.
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| Submission date |
Oct 22, 2018 |
| Last update date |
May 13, 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 |
GPL21290 |
| Series (2) |
| GSE104408 |
Aging Human Hematopoietic Stem Cells Manifest Profound Epigenetic Reprogramming of Enhancers That May Predispose to Leukemia |
| GSE121560 |
Aging Human Hematopoietic Stem Cells Manifest Profound Epigenetic Reprogramming of Enhancers That May Predispose to Leukemia (RNA-seq of KLF6 KO) |
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| Relations |
| BioSample |
SAMN10266302 |
| SRA |
SRX4910778 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3439218_AR010.txt.gz |
226.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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