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Library Info Page
| Library Name: | NIA Human H1 Embryonic Stem Cell cDNA Library (Long) |
| Organism: | Homo sapiens |
| UniGene Library ID: | 13870 |
| Tissue Description: | Embryonic stem cell line H1, undifferentiated, human |
| Library Keywords: | normal, cell line, long-transcript enriched, blastocyst, EST, embryonic stem cell, MGC, embryonic tissue, male, H1, WA01, plasmid vector, oligo-dT primed |
| #Clones Generated to Date: | 9984 |
| #Sequences Generated to Date: | 6300 |
| Library Preparation Details |
| Description: | This is a long-transcript enriched cDNA library (Genome Res. 11: 1553-1558 (2001). [PMID: 11544199]) from WA01 cell line . Undifferentiated human ES cell line WA01/H1 was obtained from WiCell Research Institute, Inc., Madison, Wi, cultured according to their instructions, on MEF feeders. They formed round colonies with defined edges and were positive for alkaline phosphatase, SSEA-4, OCT3, OCT4, REX1, UTF, TERT, SOX2, CX43 and CX45. They are negative for GATA2, GATA4, PDX1, NCAM, MSX1, FLT3, SSEA-1, TUBB3, NES, GFAP, and EOMES. When confluent (18-10 days after plating), the ES cells from 4 X 6cm dishes were treated with 1 mg/ml collagenase, type IV (Invitrogen/GIBCO) for 5-10 min and gently scraped off with 5 ml pipette. RNA was purified with TRIzol Reagent from Invitrogen. Protocol ref: Genome Res. 11: 1553-1558 (2001). [PMID:11544199]) Double-stranded cDNAs were synthesized with an Oligo(dT) primer [Invitrogen: 5'-pGACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTT-3'] from 3.4g of total RNA, treated with T4 DNA polymerase, and purified by ethanol-precipitation. The cDNAs were ligated to Lone-linker LL-Sal4, purified by phenol/chloroform extraction, and separated from free linkers by Centricon-100 column. Then, the cDNAs were amplified by long-range high fidelity PCR using Ex Taq polymerase (Takara) with a primer Sal4-S for 25 cycles. The products were purified by phenol/chloroform extraction and Centricon-100 column. The cDNAs were digested with SalI and NotI enzymes and cloned into SalI/NotI site of pCMV-SPORT6 plasmid vector. The average insert size is about 3.6kb. |
| R. Site1: | NotI |
| R. Site2: | SalI |
| Lab Host: | DH10B (T1 phage-resistant) |
| Vector: | pCMV-Sport6 |
| Vector Type: | plasmid (ampicillin-resistant) |
| Tissue Supplier: | Irene Ginis and Mahendra Rao, NIA |
| Library Producer: | Yulan Piao, Irene Ginis, Mahendra Rao and Minoru Ko (National Institute on Aging, NIH: http://lgsun.grc.nia.nih.gov/cDNA/) |
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