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| Status |
Public on Aug 04, 2011 |
| Title |
RNAseq-Fusion |
| Sample type |
SRA |
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| Source name |
RNAseq from RNA extracted from fused cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Differentiated fused HEF19 and FL24 cells
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| Growth protocol |
Two step liquid culture for differentiatioin of fused cells
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| Extracted molecule |
total RNA |
| Extraction protocol |
Direct lysis of cells, RT, second strand synthesis, cDNA amplification, RNeasy column purification of a single colony of about 100 cells originating from a single fused cell. All molecules longer than 500 bp.
We constructed a cDNA library from total RNA from a single colony of hybrid cells (0.1-1ng) as described elsewhere (Kurimoto, Yabuta et al. 2007; Tang, Barbacioru et al. 2009) with some modifications. We modified primers used for reverse transcription and second strand cDNA synthesis to include a rare-cutter restriction site (BstU1) between unique sequences (UP1 and UP2) and poly (T) fragments of the primers. This allowed us to eliminate UP1 and UP2 anchor sequences from the cDNA library which reduced a number of non-specific reads in RNA-seq. UP1_BstU1 primer: ATA TGG ATC CGG CGC GCC GTC GAC CGC GTT TTT TTT TTT TTT TTT TTT TTT T; UP2_BstU2 primer: ATA TCT CGA GGG CGC GCC GGA TCC CGC GTT TTT TTT TTT TTT TTT TTT TTT T. We increased time of the reverse transcription to 1 hour. Second-strand synthesis was conducted in a two-cycle PCR reaction (95 °C for 2 min, 50 °C for 3 min, 72 °C for 6 min). cDNA amplification was conducted with UP1_BstU1 and UP1_BstU2 primers using 25-cycle PCR reaction (95 °C for 30 sec, 68 °C for 1 min, 72 °C for 6 min plus 6 sec for each consecutive cycle). Amplified cDNA was restricted with the BstU1 endonuclease and purified with Qiaquick PCR purification kit to remove UP1 and UP2 anchor sequences. Kurimoto, K., Y. Yabuta, et al. (2007). "Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis." Nat Protoc 2(3): 739-752. Tang, F., C. Barbacioru, et al. (2009). "mRNA-Seq whole-transcriptome analysis of a single cell." Nat Methods 6(5): 377-382.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer |
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| Data processing |
Sequences were aligned against hg18 human genome using SOAP aligner. All reads mapping with two or fewer mismatches were retained.
SNPs were called using SOAPsnp tool, on aligned sequences
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| Submission date |
Aug 04, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Vladislav M Sandler |
| E-mail(s) |
[email protected]
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| Organization name |
Weill Cornell Medical College
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| Department |
Genetic Medicine
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| Street address |
1300 York Ave.
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10021 |
| Country |
USA |
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| Platform ID |
GPL9052 |
| Series (2) |
| GSE23415 |
Reprogramming of Human Fibroblasts into Hematopoietic Progenitors by Fusion With Human Fetal Liver CD34+ Cells (RNA-Seq) |
| GSE23416 |
Reprogramming of Human Fibroblasts into Hematopoietic Progenitors by Fusion With Human Fetal Liver CD34+ Cells |
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| Relations |
| SRA |
SRX025363 |
| BioSample |
SAMN00031775 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM574244_Sandler-RNAseq-Fusion-SOAPalignment-Hg18.bed.gz |
10.0 Mb |
(ftp)(http) |
BED |
| GSM574244_Sandler-RNAseq-Fusion-SOAPsnp-Hg18.txt.gz |
436.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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