BioSample

Mouse cDNA project by the Laboratory of Genetics, National Institute on Aging (NIA), Intramural Research Program, NIH (http://lgsun.grc.nia.nih.gov/cDNA).FACS-sorted Germinal Center B cells were provided by Drs. Richard Hodes, Emily Klotz (National Institute on Aging and National Cancer Institute, USA) and Garnett Kelsoe (Duke University, USA). Double-stranded cDNAs were synthesized from 0.46 ug of total RNA with an Oligo(dT) primer [Invitrogen: 5'-pGACTAGTTCTAGATCGCGAGCGGCCGCCCTTTTTTTTTTTTTTT-3'], treated with T4 DNA polymerase, and purified by ethanol-precipitation. The cDNAs were ligated to Lone-linker LL-Sal3 (Ref. Development 127: 1737-1749 (2000) [PMID: 10725249]), purified by phenol/chloroform, and separated from free linkers by Centricon 100. Then, cDNAs were amplified by long-range high fidelity PCR using Ex Taq polymerase (Takara) and purified by phenol/chloroform, followed by Centricon 100 purification. The cDNAs were digested with SalI and NotI enzymes and cloned into SalI/NotI site of pSPORT1 plasmid vector. The DH10B E. coli host was transformed with the ligation mixture by the standard chemical method. The average insert size is 1.2 kb. The library was constructed by Yulan Piao (NIA).