GSM8020686: MuSCs, total control, input rep1; Mus musculus; ChIP-Seq
Around 1 × 10^7 cells were col- lected, washed with PBS and resuspended in 250ul of ice- cold Douncing buffer (10 mM Tris-HCl (pH 7.5), 4 mM MgCl2 , 1 mM CaCl2 , 0.2% Triton X100), followed by ho- mogenization with syringe with 26 gauge X1/2 needles (BD Microlance; Cat#: BD 303800). To obtain mononu- cleosomes, cells were digested with 1.25 ul of micrococ- cal nuclease (Mnase) (0.5 U/ul; Sigma-Aldrich; Cat#: N3755-500UN) for 5 min at 37C with 600 rpm. The reaction was stopped with 10 mM EDTA, pH 8. Nu- clei were swollen to release chromatin after addition of Hypotonization buffer (0.2 mM EDTA (pH 8), FPIC (Fast Protease Inhibitors cocktail; Sigma-Aldrich; Cat#: S8830-20TAB), DTT). Chromatin corresponding to 8 ug of DNA was incubated with anti-H3K9me3 (Diagenode; Cat#: C15410193), anti-H3K27me3 (Diagenode; Cat#: C15410069), anti-H3K27ac (Diagenode; Cat#: C15410196) or anti-H3K4me3 (Diagenode; Cat#: C15410003) antibod- ies overnight at 4C. The DNA protein antibody com-plexes were captured by DiaMag protein A-coated mag- netic beads (Diagenode; Cat#: C03010020) by incubating at 4C for 2 h. Magnetic beads were then washed with Low- Salt buffer (20 mM Tris?HCl (pH 8), 0.1% SDS, 1% Tri- ton X100, 2 mM EDTA (pH 8), 150 mM NaCI and FPIC) and High-Salt buffer (20 mM Tris?HCl (pH 8), 0.1% SDS, 1% Triton X100, 2 mM EDTA (pH 8), 500 mM NaCI and FPIC). DNA was eluted with TE buffer (100 mM Tris-HCl (pH 8), 1% SDS, 1 mM EDTA (pH 8)) from the beads overnight, inputs corresponding to 1% of IP were treated as well, followed by incubation with 1 _x0002_l of RNase A (10 ug/ml; Sigma-Aldrich; Cat#: R4642) for 1 h. DNA was pu- rified using MinElute PCR Purification Kit (Qiagen; Cat#: 28004). Libraries were prepared using the MicroPlex V2 library preparation Kit (Diagenode; Cat#: C05010012), following the manufacturer's instructions, 10ul of DNA was used as starting material. After end repair of the double-stranded DNA templates, cleavable stem-loop adaptors were ligated, and then adaptor enriched DNA was amplified with 10 PCR cycles to add Illumina compatible indexes. The li- braries were then purified with the Agencourt AMPure XP bead-based purification system. Final libraries were quan- tified with Qubit dsDNA HS assay (Thermo Fisher; Cat#: Q32851), and size distribution was monitored by capillary electrophoresis on Agilent 2100 Bioanalyzer using DNA High Sensitivity HS Kit. Libraries were then normalized to 10 pM and pooled before sequencing. Libraries were sequenced on an Illumina Nextseq 500 in a paired-end mode in two independent biological replicates at Platform GENOM IC Institute Cochin.