fraction43 sample
Homo sapiens
"A long-insert fosmid clone library was constructed using genomic DNA from individual GM20847 as previously described (Raymond CK et al, 2005), with minor modifications as follows. A lymphoblastoid cell line for the individual of interest (GM20847, Coriell) was cultured according to standard techniques. High molecular weight genomic DNA (HMW gDNA) was isolated from ~1.4e7 cells using the Gentra Puregene Cell Kit (QIAGEN, 158388). 8 ug of HMW gDNA was sheared using a Bioruptor sonicator (Diagenode) in 60 uL of TE8 for 20 seconds on the ""medium"" setting. The sheared DNA was electrophoresed in a 1% Ultrapure agarose gel (Invitrogen, 16500100) and using a Bio-Rad CHEF-DR II Pulsed Field Gel Electrophoresis apparatus at 160V with an initial switching time (""Initial A time"") of 1 second, a final switching time (""Final A time"") of 6 seconds, and a run-time of 20 hours. The gel was run for ~16 hours, stained with Ethidium Bromide according to standard practice, the lanes containing ladder were removed and imaged, and the gel was reconstructed so as to avoid exposure of the sample DNA to UV light. A band corresponding to ~35-45 kb was excised from the gel, DNA was electroeluted from the gel as previously described (Strong SJ et al, 1997), and purified by ethanol precipitation. Size-selected DNA was end-repaired using the End-IT kit (Epicentre, ER0720), purified by phenol-chloroform extraction and ethanol precipitation, and blunt-end ligated to prelinearized pCC1FOS (Epicentre, Cat. No. CCFOS110) according to manufacturer's specifications (using ~125 ng of input DNA and a 10:1 vector:insert molar ratio). Packaging reaction using MaxPlax Lambda packaging extract was performed according to manufacturer's instructions except that chloroform was not added after diluting using the Phage Dilution Buffer. TransforMax EPI300 T1R Chemically Competent E. coli (Epicentre, Cat. No. C300C105) were expanded for bulk infection in 22ml LB with 10mM MgSO4 and 0.2% maltose in a shaking incubator for 14 hours at 37?C. 16ml LB with 10mM MgSO4 and 0.2% Maltose was inoculated with 1ml of the expanded culture and grown with shaking at 250rpm and 37?C until OD600 ~ 0.7. Cells were then pelleted by centrifugation at 2000rpm and 4?C for 10 minutes. Pellet was resuspended in 9.6ml 10mM MgSO4 and infected by room temperature incubation with 240ul packaged phage. After 30 min, 9.6 ml LB was added and cells were incubated for 30 min at 37C with shaking at 175rpm. Total library complexity of ~2.5x10^6 clones was estimated by plating."