title: CUT&Tag chromatin profiling of H2AK119Ub, H3K27me3, H3K27ac and H3K4me3 histone in  MC38 colon carcinoma cell line
design: CUT&Tag chromatin profiling of H2AK119Ub, H3K27me3, H3K27ac and H3K4me3 histone in  MC38 colon carcinoma cell line
library_construction_protocol: Tumor cells expressing BFP fluorescent protein were isolated from Rag2-/- mice, Tumor Dissociation Kit (Miltenyi,130-096-730) was used to dissociate the tumor tissues into single cells as the manufacturer's instructions. ACK lysis buffer (Gibco,492-01) was used to remove the red blood cell. Then Dead Cell Removal kit (Miltenyi,130-090-101) was used to remove dead cells by column in MACS Separator,then Then the tumor cells were resuspended with Facs buffer and BFP+ tumor were sorted at 4℃. CUT&Tag experiment was  performed according to the reaction system recommended by Vazyme #TD901-2 follow the instructions. To stop the fragmentation reaction,  10 μl 0.5M EDTA,3 μl 10% SDS and 2.5 μl 20 mg/ml Proteinase K were incubated with mixture overnight at 37℃. 150 μl Tris saturated phenol and 150 μl chloroform were added and centrifuge for 5 min at 16,000 g to collect the supernatant. Add 300 μl chloroform and centrifuge to collect the supernatant, then which was added to 750 μl 100% ethanol in tube on ice, and centrifuge for 15 min at 1,6000 g at 4℃to collect the DNA. Last sample was washed using 100% ethanol, and dissolved in double-distilled water. For library construction, set up the PCR reaction as follows: for a total volume of 50 μL, add 24 μL of DNA,5 μL of ddH2O,10 μL of 5 × TAB buffer, 5μL P5 primer X, 5 μL P7 primer X, and 1 μL of TAE. Mix gently and spin briefly. Then PCR amplification program were carried out,
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-10585_2:C-H2AK119ub_p