GSM2193298: MN.WT; Mus musculus; OTHER
50 million cells were trypsinized and crosslinked in1% formaldehyde for 10 min at room  temperature. The primary restriction enzyme used was HinDIII. Ligation and xLink reversal was performed and DNA purified for prep of 5C library. 5C primers were annealed at 48C for 16hrs atop 2 biological replicates of 3C libraries from each sample. 1fmol of each primer was used in the annealing reaction with 1ug of 3C template and 500ng of salmon sperm DNA. 16 separate annealing reactions were performed per sample, along with control reactions with individual components removed. Forward and reverse primers that annealed to adjacent regions of the 3C template were ligated with 10U of Taq ligase for 60' at 48C. Successfully ligated forward-reverse primer pairs were then amplified in 5 separate PCR reactions per annealing reaction, using primers specific to the T7 and T3 overhangs. PCR reactions from the equivalent initial sample were then pooled, purified and run on a gel to ensure the control reactions did not show an amplification product. Libraries were then generated from the purified PCR product as described above to allow for deep sequencing.
GEO Accession
GSM2193298