GSM1399403: 21 months rep2; Mus musculus; MNase-Seq
Digestion was performed on snap-frozen liver. 100 mg of tissue was pulverized (Covaris cryoPrep impactor), and nuclei were purified using a sucrose gradient. The nuclei were digested with MNase (Roche catalog number 10107921001) at 37ºC for 12 minutes. Several titrations of MNase enzyme were tested. Samples that were well-digested, with most of the input as a mononucleosome (150 bp) and a slight band of a dinucleosome (300 bp) visible on a BioAnalyzer trace, to prevent over-digestion, were used for subsequent library preparation. Libraries were prepared according to Illumina's instructions. Briefly, DNAs were blunted with a combination of T4 DNA polymerase, Klenow polymerase, and T4 PNK, then a single 3′-end “A” base was added using Klenow exo (3′-to-5′ exo minus). Broad custom adapters were then ligated to the ends of the modified DNA.  After adapter ligation DNA was PCR amplified with Broad custom primers for 16 cycles and library fragments of ~280 bp (~150-bp mononucleosome insert plus ~130-bp adaptor and PCR primer sequences) were  isolated using Pippin Prep agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on Illumina Hiseq 2000 following the manufacturer's protocols.
GEO
GSM1399403
GEO Accession
GSM1399403