|
| Status |
Public on Jan 01, 2019 |
| Title |
CB-HSPC-RNAseq-r1 |
| Sample type |
SRA |
| |
|
| Source name |
hematopoietic stem/progenitor cell from cord blood
|
| Organism |
Homo sapiens |
| Characteristics |
cell surface marker: CD34+
cell type: hematopoietic stem/progenitor cell from cord blood
|
| Treatment protocol |
Wnt inhibitors were added on day 6 of the differentiation protocol. Half of the medium was refreshed every other day with inhibitors until cell harvest
|
| Growth protocol |
iPSCs (passage 11) and ESCs cultured on MEF feeders in human ESC medium were manually dissociated and plated on 8 days old overgrown OP9 feeders to start differentiation. Cells were co-cultured with the OP9 feeders for 12-14 days in OP9 differentiation medium (Alpha MEM containing 10% FBS, 100 μM monothioglycerol and 50 μg/ml ascorbic acid) as described by Vodyanik, M. A. & Slukvin, II. (Curr Protoc Cell Biol 2007). Differentiated cells were dissociated with collagenase and Accutase (Innovative Cell Technologies) and subjected to cell sorting, RNA isolation and transposition assay. Cord blood CD34+ cells were isolated from donated cord blood units by immnuselection with CD34 magnetic microbeads (Miltenyi biotec).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted with Qiagen RNeasy micro kit. ATAC-seq was carried out according to Buenrostro et al (Nat. Methods, 2013).DNA was purified using Zymo ChIP DNA Clean & Concentrator kit (Zymo Research).
The Agilent TapeStation or Bioanalyzer was used to determine RNA integrity (RIN) numbers prior to library preparation. Stranded mRNA-Seq libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit according to the manufacturer’s instructions (Illumina). Briefly, RNA with polyA tail was isolated using poly-T oligos conjugated to magnetic beads. mRNA was then fragmented and reverse-transcribed into cDNA. dUTPs were incorporated, followed by second strand cDNA synthesis. dUTP-incorporated second strand was not amplified. cDNA was then end-repaired, index adapter-ligated and PCR amplified. Purification of nucleic acids after each enzymatic steps was achieved by using AMPure XP beads (Beckman Coulter).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
RNA-Seq of hematopoietic progenitor cells from cord blood
fpkm.txt
|
| Data processing |
Illumina Casava v1.8.2 software used for basecalling.
For RNA-Seq, paired-end reads were aligned to the human genome (hg19) with STAR (v2.2.0c) with default parameters. For ATAC-Seq, single end reads were aligned to the human genome (hg19) using Bowtie2 (v2.2.5) with default parameters. Only uniquely alignable reads were used for downstream analysis.
For RNA-Seq, Fragments Per Kilobase of exon per milion mapped reads were calculated using HOMER (http://homer.salk.edu/homer/) across annotated gene exons (RefSeq). ATAC-Seq reads were analyzed for peaks using HOMER by treated the experiments as default ChIP-Seq experiments.
Genome_build: hg19
Supplementary_files_format_and_content: Tab delimited text file, Columns: (1) RefSeq acc (2) chr (3) start (4) end (5) strand (6) length (7) copies (8) annotation (9-14) FPKM.
|
| |
|
| Submission date |
Jun 16, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Christopher Benner |
| E-mail(s) |
cbenner@ucsd.edu
|
| Organization name |
University of California, San Diego (UCSD)
|
| Department |
Medicine
|
| Street address |
9500 Gilman Dr. MC 0640
|
| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92093-0640 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE69932 |
Comparison of the transcriptome and chromatin state between human cord blood HSC and human iPSC derived hematopoietic progenitor using next-generation sequencing |
|
| Relations |
| BioSample |
SAMN03776953 |
| SRA |
SRX1060091 |
