TLA sequencing of Berkeley splenocytes, primer 4 (HBB)
Splenocytes were obtained from 8 week-old mice by homogenizing splenic tissue, passing through a 40 m filter and rinsing with PBS + 10% FBS. RBCs were lysed and the remaining mononuclear cells were cryopreserved in PBS with 10% DMSO and 10% FCS. Cells were formaldehyde cross-linked, digested into approximately. 0.2 kb fragments by NlaIII and ligated to form larger circular DNA segments that include regions of neighboring DNA. The circular DNA was then de- crosslinked  and digested with NspI to form 2kb fragments of DNA.31 PCR primer pairs were designed to each of the three transgenic fragments and used to selectively amplify and sequence regions of DNA that were near to the transgenic fragments of interest within the Berkeley mouse genome.