title: GSM1835354: S3; Homo sapiens; MNase-Seq
library_construction_protocol: Sperm is washed with PBS for twice and resuspended in a lysis buffer contaning NP-40 and sodium deoxycholate. Mnase was added to digest genomic DNA without the protection of histone. Sooluble chromatin fraction can be isolated upon centrifugation. DNA is obtained by phenol/chroloform extraction after treatment with RnaseA and proteinase K. DNA is size-selected by 5% agrose-gel electrophoresis to ensure the use of mononuclesomal DNA(150bp) in the preparation of sequecing libraries. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~150 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.