title: GSM3325487: BG3_GST; Drosophila melanogaster; MNase-Seq
library_construction_protocol: 9 x 106 cells per condition were washed in 10 ml cold PBS and resuspended in 1 ml TM2 buffer (10 mM Tris-HCl pH 7.5, 2 mM MgCl2, 1 mM PMSF). The cells were incubated 1 min on ice and NP-40 added to 1.5 %, gently vortexed, and again incubated on ice for 10 min for lysis and release of the nuclei. After a 10 min centrifugation at 93 x g and 4°C the nuclei were resuspended in 600 ml of TM2 buffer with 2 mM CaCl2 and distributed into three 200 ml aliquots. Those were treated with 2.3/5.9/11.7 x 10-3 units of MNase for 20 min at 37°C. The reaction was then quenched by adding 5 ml 0.5 M EDTA and 100 mg RNase A, followed by a 20 min incubation at 37°C. Proteins were digested away at 50°C for 2 h after the addition of 12 ml 10% SDS and 200 mg Proteinase K. The DNA was then purified by Phenol/Chloroform extraction, precipitated in 2.5 V EtOH, 1/10 V NaOAC 3M and 20 mg glycogen and run on a 1.2% agarose gel in 0.5x TAE buffer. The samples with the best digestion degree (predominantly nucleosomes, achieved with 11.7 x 10-3 U of MNase) were chosen and the mononucleosomal bands cut out and purified (NucleoSpin Gel and PCR Clean-up, Macherey-Nagel). The purified DNA was then used for Next-Generation-Sequencing library preparation. Libraries were prepared on 10 ng of DNA with the iDeal library preparation kit (Diagenode). PCR amplification was monitored by quantifying amplified libraries.
attributes.tag: GEO Accession
attributes.value: GSM3325487