title: GSM8833940: EZH2KO-12-H3K27me3 rep2; Mus musculus; OTHER
library_name: GSM8833940
library_construction_protocol: Cells were collected for CUT&TAG libraries construction without extraction. CUT&TAG libraries were prepared using the Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro (Vazyme Biotech, #TD904). Briefly, 1 × 105 cells were washed with 500 μl wash buffer and centrifuged at 200×g for 5 min at room temperature. Cell pellets were resuspended with 100 μl wash buffer. A total of 10 μl concanavalin A-coated magnetic beads were washed twice with 100 μl binding buffer and then added to the cell tubes and incubated at room temperature for 10 min. After removing the supernatant, the bead-bound cells were resuspended with 50 μl antibody buffer containing primary antibody (Dilute according to instructions). After incubation overnight at 4°C, the primary antibody was carefully discarded and anti-rabbit IgG (Vazyme, Ab207, 1:100) diluted with 50 μl Dig-wash buffer was added to the cells. The cells were then incubated with rotation at room temperature for 1 h. The cells were then washed gently with 200 μl Dig-wash buffer three times, and 2 μl pA/G–Tnp together with 98 μl Dig-300 buffer was added to the samples. After incubating at room temperature for 1 h, the samples were washed gently with 200 μl Dig-300 buffer three times. Then, 10 μl 5 × TTBL mixed with 40 μl Dig-300 buffer was added to each sample and the samples were incubated at 37 °C for 1 h. The interactions were quenched by adding 2 μl 10% SDS and incubating the samples at 55°C for 10 min. The supernatant was transfered to a new tube, and added 50 μl DNA Extract beads to  purify the DNA. For library amplification, 15 μl of purified DNA was mixed with 25 μl of 2× CAM, along with 5 μl of uniquely barcoded i5 and i7 primers from the TruePrep Index Kit V2 for Illumina (Vazyme Biotech, # TD202). A total volume of 50 μl of sample was placed in a BioRAD cycler (BIO-RAD, #T100) using the following program: 72 °C for 3 min; 98 °C for 3 min; 12 cycles of 98 °C for 10 s, 60 °C for 5 s, and 72°C for 1 min; and holding at 14 °C. To purify the PCR products, 100 μl volumes of VAHTS DNA Clean Beads (Vazyme Biotech, #N411) were added and incubated at room temperature for 5 min. The beads were washed twice with 200 μl fresh 80% ethanol and eluted in 25 μl elution buffer. All CUT&Tag libraries were sequenced by BerryBenomics using the Illumina NovaSeq 6000 platform in PE150 mode (BerryBenomics, Beijing, China).
identifiers.namespace: GEO
identifiers.id: GSM8833940_r1