GSM4175622: Rep5_ec_none_Chd1_40; Escherichia coli; MNase-Seq
Remodeling was started by adding 10 µl SGD chromatin and terminated by adding 0.8 U apyrase followed by incubation at 30 °C for 30 minutes. For library preparation, approximately 10 - 50 ng of mononucleosomal DNA was incubated with 1.25 U Taq polymerase (NEB), 3.75 U T4 DNA polymerase (NEB) and 12.5 U T4-PNK (NEB) in 1x ligation buffer (B0202S, NEB) for 15 minutes at 12 °C, 15 minutes at 37 °C and 20 minutes at 72 °C. To ligate NEBNext Adaptors (0.75 µM final concentration, from NEBNext Multiplex Oligos Kit) to the DNA, samples were incubated with T4 DNA ligase (NEB) at 25 °C  for 15 minutes, followed by incubation with 2 U USER enzyme (NEB) for 10 minutes at 37°C. Fragments were purified using 2 volumes AMPure XP beads (Beckman Coulter) and amplified for 8-10 cycles using NEBNext Multiplex Oligos, Phusion High-Fidelity DNA Polymerase (1 U, NEB), deoxynucleotide solution mix (dNTP, 2.5 mM, NEB) and Phusion HF Buffer (1x, NEB). The following protocol was applied for amplification: 98 °C for 30 s, 98°C for 10s, 65 °C for 30s, 72 °C for 30s with a final amplification step at 72 °C for 5 minutes. The Qubit dsDNA HS Assay Kit (Invitrogen) was used to assess the DNA content of 1 µL PCR reaction. DNA was purified from agarose gels via PureLink Quick Gel Extraction Kit, measured again with Qubit dsDNA HS Assay Kit and diluted to final concentration of 10 nM (calculation based on the assumption that the DNA fragment length is 272 bp, i. e., 147 bp nucleosomal DNA and 122 bp sequencing adaptor). Diluted samples were pooled according to sequencing reads (~6 Mio reads/ sample). The final pool was quantified by BioAnalyzer (Agilent) and analyzed on an Illumina HiSeq 1500 in the 50 bp single-end mode (Laboratory for Functional Genome Analysis, LAFUGA, LMU Munich).
GEO
GSM4175622
GEO Accession
GSM4175622