title: NextSeq 500 paired end sequencing; DNA- and RNA-seq to study the impact of 5-formylcytosine on nucleosome organization and gene expression in embryonic mouse tissues and stem cells
design: DNA- and RNA-seq to study the impact of 5-formylcytosine on nucleosome organization and gene expression in embryonic mouse tissues and stem cells
library_name: h3_reduced_2_p
library_construction_protocol: TDG -/- mouse embryonic stem cells were cultured on gelatinized plates in DMEM medium supplemented with 15% fetal bovine serum, 1% Penicillin Streptomycin, 1% MEM non-essential amino acids, 1% Glutamax, 50 μm β-mercaptoethanol and 103 U LIF, in normoxic conditions at 37°C. Tissues were collected from timed matings with the day of the copulation plug as E0.5. Embryos were dissected into hindbrain and heart. Nuclei (mESC) were treated with NaCNBH3 (80mM) in PBS for 3h at 37°C. Nuclei were sonicated for 8 min on Covaris M220 using the truChIP Chromatin Shearing Kit (Covaris) following the manufactures guidelined. Protein denaturation was achieved using binding buffer containing a chaotropic agent (from Genejet PCR purification kit, Thermofisher) followed by 4x 450 μL water washes in the Amicon Ultra-Spin column (30kDa cutoff) to obtain sheared DNA fragments. DNA libraries were obtained by end repair, A-tailing and adapter ligation (NEBNext Ultra II DNA library Prep kit) following the manufacturer's guideline.  For ChIP, 100 μL dynabeads M-280 sheep anti-rabbit IgG were washed and preincubated with 10 μg antibodies (Upstate 06597, ab18255, ab1791 and ab8227) in PBS/BSA for 3 h at 4°C. Beads were subsequently washed with PBS/BSA and DNA libraries together with 1 ug salmon sperm DNA were added to the beads and incubated overnight at 4°C. Beads were then washed (5 min rotation at room temperature) 6x with 500 μL LiCl buffer (100 mM TRIS pH7.4, 500 mM LiCl, 1% NP40, 1% sodium deoxycholate) and 3x tube changes. For elution, beads were incubated (15 min rotation at room temperature) with 2x 100 μL elution buffer (0.1 NaHCO3, 1% SDS). To the combined 200 μL supernatant were then added 8 μL of 5 M NaCl, 4 μL of TRIS pH 7.4, 2 μL of 0.5 M EDTA and 1 μL proteinase K (10 mg/mL), incubated at 45°C for 1 h and purified using the Genejet PCR purification kit.
attributes.tag: Experimental Factor: protocol
attributes.value: ChIP-seq
attributes.tag: Experimental Factor: organism
attributes.value: Mus musculus
attributes.tag: Experimental Factor: genotype
attributes.value: Tdg knockout
attributes.tag: Experimental Factor: organism part
attributes.value: inner cell mass
attributes.tag: Experimental Factor: compound
attributes.value: sodium cyanoborohydride
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: anti-H3