GSM2055466: Time Point 3 - MNaseSeq; Saccharomyces cerevisiae; MNase-Seq
chromatin was formaldehyde crosslinked prior to nuclease digestion (1% formaldehyde with gentle shaking at room temp for 10 minutes) 500ng from each sample was incubated with 10U of PNK in a reaction containing 1X T4 Ligase Buffer, and 1mM dNTPs for 30 minutes at 37˚C (Total volume = 20ul).. 3U of T4 Pol was then added to each sample and incubation was allowed to proceed for 15 minutes at 12˚C. Subsequently, 1ul of 0.5M EDTA was added to inhibit the activity of T4 Polymerase and the reaction was killed by incubation at 75˚C for 20 minutes. 1.8 volumes of Ampure beads was added to each reaction to purify the nucleosomal DNA and remove the PNK and T4 Pol. DNA was resuspended in 15ul of TE (pH 8). dA tailing reactions of end cleaned DNA included 1X Taq Buffer, 1mM dNTPs, and 5U of Taq Polymerase (Total volume = 20ul), and were incubated at 72˚C for 30 minutes. Reactions were then again cleaned with 1.8 volumes of Ampure beads and resuspended in 17.5ul TE. To the dA tailed products we then ligated dT tailed hairpin adaptors purchased from NEB (Catalog #E7500L). Ligation reactions included the 17.5ul of nucleosomal DNA, 1X T4 Ligase Buffer, and 400U of T4 DNA Ligase (Total volume = 25ul), and were allowed to proceed at room temperature for 1 hour. To remove unligated adaptors and clean up the reaction, 1.8 volumes of Ampure beads were added and the adaptor ligated nucleosomal DNA was subsequently resuspended in 25ul of TE. Half of this product was then used in a reaction which served to both amplify and barcode each sample. PCR reactions included the adaptor ligated substrates, 1uM Universal Oligo, 1mM Barcode Addition Oligo, 3U of NEB USER enzyme, and 1X KAPA Hot Start Polymerase Mix (Final volume = 50ul). Libraries were amplified for 10 cycles of PCR. PCR reactions were purified with 1.8 volumes of Ampure beads and resuspended in 20ul of TE. Products were then run on a 1.4% agarose gel and monosomal bands were excised and gel purified.  The MNase digested samples were sequenced on the Illumina HiSeq platform, and the paired-end reads were mapped back to the S. cerevisiae genome using the Bowtie 2 software.
GEO Accession
GSM2055466