GSM5380052: CUT&Tag_Crotonate-H3K27me3; Mus musculus; OTHER
CUT&Tag assays were performed according the instructions of HyperactiveTM In-Situ ChIP Library Prep Kit for Illumina (TD-901, Vazyme). Briefly, 1 × 105 NSPCs were washed with 500 μl Wash Buffer and entrifuged at 600 × g for 3 min at room temperature and then resuspend in 100 μL 1 × Wash Buffer. ConA beads were washed twice with Binding Buffer. Next, 10 μL of washed ConA beads were added into NSPCs and incubated at room temperature with slow rotation for 10 min. Bead-bound NSPCs were resuspended in 50 μL of Antibody Buffer. Then, 1 μg of primary antibody (H3K4me3 or H3K27me3) or normal rabbit IgG was added and incubated at room temperature with slow rotation for 2 h. The primary antibody was removed using a magnet stand. The secondary antibody (0.5 μL) (Goat anti-Rabbit IgG H&L (ab6702, abcam)) was diluted in 50 μL of Dig-wash Buffer and NSPCs were incubated at room temperature with slow rotation for 1 h. NSPCs were washed three times with Dig-wash Buffer to remove unbound antibodies. Next, NSPC were incubated with 0.44 μM The Hyperactive pG-Tn5 Transposon diluted in 100 uL Dig-300 Buffer at room temperature with slow rotation for 1 h, and subsequently washed three times with Dig-300 Buffer. NSPCs were then resuspended in 300 μl of Tagmentation Buffer at 37 °C without rotation for 1 h. To terminate tagmentation, 10 μL of 0.5 M EDTA, 3 μL of 10% SDS and 2.5 μL of 20 mg/mL Proteinase K were added to 300 μl of sample and incubated overnight at 50 °C without rotation. DNA was purified using phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation. 1 × cOmpleteTM, EDTA-free Protease Inhibitor Cocktail (04693132001, Roche) was pre-added in all buffers mentioned above. For library amplification, 24 μL of DNA was mixed with 1 μL of TAE of HyperactiveTM In-Situ ChIP Library Prep Kit for Illumina (TD-901, Vazyme), 10 μL of 5 × TAB and 5 μL of ddH2O, as well as 5 μL of uniquely barcoded i5 and i7 primers from TruePrep® Index Kit V2 for Illumina (TD-202, Vazyme). A total volume of 50 μL of sample was placed in a Thermocycler using the following program: 72 °C for 3 min; 98 °C for 30 s; 16 cycles of 98 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s; 72 °C for 5 min and hold at 4 °C. To purify the PCR products, 1.2× volumes of VAHTS® DNA Clean Beads (N411, Vazyme) were added and incubated at room temperature for 10 min. Libraries were washed twice with 80% ethanol and eluted in 22 μL of ddH2O, and were sequenced on an Illumina NovaSeq 6000 platform and 150-bp paired-end reads were generated.