GSM3069853: VSMC_P10_MNase; Mus musculus; MNase-Seq
RNA-Seq: Total RNA was extracted from cells using the GeneJet RNA Purification Kit (Thermo Scientific) 4 days after infection. One ?g of RNA was used for generation of sequencing library using the TruSeq RNA Library Prep Kit V2 (Illumina) according to the manufacturerЎЇs instructions. ChIP-Seq: Mouse VSMCs were fixed using 1% formaldehyde for 10 min, and 0.125 M glycine was added to stop fixation. Cells were harvested, and DNA was fragmented to 300ЁC500 bp by sonication with Covaris S220 sonicator. Immunoprecipitation was performed with antibodies conjugated to Dynabeads Protein G beads (Life Technology). ChIP DNA was eluted, reverse cross-linked, extracted by phenol/chloroform, and precipitated. MNase-Seq: Cultured mouse vascular smooth muscle cells were trypsinized and washed twice with PBS. Then, mononucleosomes were prepared using the EZ nucleosomal DNA Prep Kit (D5220, Zymo Research) according to manufacturerЎЇs instructions. Briefly, 1ЎБ106 cells were incubated with 100 ?l Nuclei Prep Buffer for 5 minutes on ice, followed by one wash with 100 ?l Atlantis Digestion Buffer. Isolated nuclei were incubated with 0.5 U Atlantis ds DNase at 42ЎгC for 20 minutes to react, and stopped by adding 5 ЎБ MN Stop Buffer. Nucleosomal DNA was purified with Zymo-SpinIIC column. To obtain mononucleosome, purified nucleosomal DNA was first incubated with 0.8ЎБ Agencourt AMPure XP beads (Beckman Coulter), and supernatant was kept. Afterwards, the collected supernatant was purified with 1.8ЎБ beads, and nucleosomal DNA was evaluated and quantitated by Agilent 2100 Bioanalyzer (Agilent).Nucleosomal sequencing library was prepared using KAPA HyperPlus Kit (Kapa Biosystems) according to manufacturerЎЇs instructions.
GEO Accession
GSM3069853