title: GSM8839223: Foxk2_KO_GM_3; Mus musculus; RNA-Seq
library_construction_protocol: For ChIP-seq, C2C12 cells (5-10.0×106) were crosslinked  with 1% formaldehyde and then quenched in a final solution at a final concentration of 0.125 M. The chromatin was extracted and digested with micrococcal nuclease to primarily generate mononucleosomes, with a minor fraction of dinucleosomes. Digestion reaction was halted by adding 8μL of a solution containing 0.2 M EDTA and 0.2 M EGTA. The chromatin was then incubated overnight at 4°C with either 2 μL of rabbit anti-FOXK2 antibody or 2 μL control rabbit IgG antibody. Protein A/G magnetic beads (Thermo Fisher) were added for an additional 4 hour incubation. The resulting immunoprecipitates were washed thoroughly to remove unbound materials, and DNA was purified according to the manufacturer's instructions using the SimpleChIP® Plus Enzymatic Chromatin IP Kit (CST). For RNA-seq, Total RNA was extracted from cells using TRIzol reagent (Invitrogen), and the RNA quantity and purity were assessed using the Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent). Only samples with an RNA integrity number (RIN) > 7.0 were selected for subsequent library construction. Paired- end (PE) RNA sequencing was performed on the Illumina NovaseqTM 6000 platform (Illumina). For ATAC-seq,   To profile open chromatin regions, a totol of 50,000 control and FOXK2 knockout C2C12 cells were washed once with cold PBS. Cells were lysed on ice for 3 minutes in 50 μL of ice-cold lysis buffer containing 10 mM Tris (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40, 0.1% Tween 20, and 0.01% Digitonin in diethylpyrocarbonate (DEPC)-treated water. Following lysis, cells were resuspended in ice-cold RBS-Wash buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween 20) and pelleted at 4 °C at 500 × g for 10 minutes. Tagmentation was performed in 1× Tagmentation buffer composed of 10 mM Tris (pH 7.4), 5 mM MgCl2, 10% DMF, 33% PBS, 0.1% Tween 20, and 0.01% Digitonin, using 100 nM Tn5 Transposase for 15 minutes at 37 °C. An equal volume of 2x Tn5 Digestion Mix (TransNGS) was added, and the tagmentated samples were purified using the QIAquick PCR Purification Kit (Qiagen). Libraries were then amplified with Illumina primers, purified again, and size-selected using 0.7× and 0.2× DNA clear beads (TransNGS). ChIP-seq, ATAC-seq,RNA-seq
identifiers.namespace: GEO
identifiers.id: GSM8839223_r1