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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 14, 2021 |
| Title |
Differentiated erythroid cells - Phase II Day 11 - Replicate 1 |
| Sample type |
SRA |
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| Source name |
Cultured erythroid cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Differentiated intermediate stage erythroid cells
time point: Day 11 of phase II of the culture
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| Treatment protocol |
No treatment was performed.
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| Growth protocol |
CD34+ HSPCs were isolated from mobilized peripheral blood of a healthy individual using a positive magnetic bead-based selection kit (StemCell Technologies, Vancouver, BC, Canada). Phase I of the culture, 5 X 10^6 purified CD34+ cells were seeded in a serum-free HSPC expansion medium composed of StemSpan SFEM-II (Stem Cell Technologies, Vancouver, Canada) supplemented with 100 U/ml penicillin,100 µg/ml streptomycin (Thermo Fisher Scientific, Inc., Grand Island, NY, USA), 2mM L-glutamine (Thermo Fisher Scientific, Inc., Grand Island, NY, USA), 100ng/ml recombinant human stem cell factor (rh SCF), 100ng/ml recombinant human fms related tyrosine kinase 3-ligand (rh Flt3-L), 20ng/ml recombinant human interleukin-6 (rh IL-6), 50ng/ml recombinant human interleukin-3 (rh IL-3) and 100ng/ml recombinant human thrombopoietin (rh TPO). After expanding the cells for 5 days in phase I of the culture, the cells were transferred to phase II of the culture in Stem Span SFEM-II medium containing 50ng/ml rh SCF, 40ng/ml recombinant human insulin growth factor-1 (rh IGF1),10ng/ml IL-3 and 3U/ml recombinant human erythropoietin (rh Epo). All cytokines were purchased from Peprotech Inc., Rocky Hill, NJ, USA. The cell density was maintained at 0.5 X 10^6 cells/ml throughout the culture, and the medium was changed every alternate day.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Total RNA Purification Kit (Norgen Biotek Corp).
Small RNA libraries were prepared NEBNext Multiplex Small RNA Library Preparation Kit (New England Biolabs, Ipswich, MA, USA) as per the manufacturer’s protocols. The products were checked for fragment size distribution (140-160bp) on TapeStation (Agilent Technologies) using High Sensitivity D1000 DNA ScreenTapes (Agilent Technologies) followed by size selection (140-160bp) on 4% E-Gel EX Agarose Gel (Thermo Fisher Scientific, Rockford, IL, USA). Prepared libraries were quantified using Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). The obtained libraries were pooled and diluted to a final optimal loading concentration before cluster amplification on the Illumina flow cell (Illumina Inc., San Diego, CA, USA). Once the cluster generation was completed, the cluster flow cell was loaded on Illumina HiSeq 2500 instrument to generate 20 million 50bp paired-end reads.
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| Library strategy |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Phase II Day 11- R1
Early stage erythroid progenitor cells from day 11 of phase II of the culture
Small RNA-seq
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| Data processing |
The bioinformatics analysis to estimate the differential expression of miRNAs was performed with The UEA small RNA Workbench (http://srna-workbench.cmp.uea.ac.uk/) using default settings allowing 1 mismatch during the alignments with the small RNA sequence lengths ranging from 16 to 26 nucleotides.
The reads were aligned with miRBase version 22.1 (https://www.mirbase.org/ftp.shtml), and normalization of the miRNA reads and differential expression were calculated using DESeq using default parameters.
Human genome hg19 reference fasta files were indexed using bowtie tool version 0.12.9.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: Processed_files_RVS_raw_reads_GEO.txt: Tab-delimited text file includes DESeq normalized counts.
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| Submission date |
Oct 11, 2021 |
| Last update date |
Oct 14, 2021 |
| Contact name |
Shaji R Velayudhan |
| E-mail(s) |
[email protected]
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| Organization name |
Christian Medical College
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| Street address |
Centre for Stem Cell Research, Christian Medical College, Bagayam
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| City |
Vellore |
| State/province |
Tamil Nadu |
| ZIP/Postal code |
632002 |
| Country |
India |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE185685 |
Small RNA analysis of human erythropoiesis |
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| Relations |
| BioSample |
SAMN22217339 |
| SRA |
SRX12572146 |
| 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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