GSM5706740: IFNβ_Flag SP100A; Homo sapiens; OTHER
CUT&Tag assay was performed using NovoNGS CUT&Tag 2.0 High-Sensitivity Kit (for Illumina) (Novoprotein scientific Inc, N259-YH01-01A) as previously described 61. In brief, 1X105 HEp-2 or HEp-2 cells transfected with Flag-Sp100A were harvested by trypsin treatment, centrifuged at 800g for 5min at RT, precipated by ConA-magnetic beads, and resuspended in 100mL Dig-wash Buffer (20mM HEPES pH7.5, 150mM NaCl, 0.5mM Spermidine, protease inhibitor cocktail, 0.05% Digitonin) containing 2mM EDTA and primary anti-Flag antibody at 1:100 dilution overnight at 4°C. Beads were washed in Dig-wash Buffer for 3 times and incubated with goat anti-mouse secondary antibody (Abcam, ab6708) for 1hr at a dilution of 1:200. After incubation, the beads were washed for 3 times in Dig-Hisalt Buffer (0.05% Digitonin, 20mM HEPES, pH7.5, 300mM NaCl, 0.5mM Spermidine, protease inhibitor cocktail). Cells were incubated with proteinA-Tn5 transposase at 37°C for 1hr and washed for 3 times in Dig-Hisalt buffer to remove unbound proteinA-Tn5. Next, cells were resuspended in 100uL Tagmentation buffer (10mM MgCl2 in Dig-Hisalt Buffer) and incubated at 37°C for 1hr. The tagmentation was terminated by adding 2.25mL of 0.5M EDTA, 2.75mL of 10% SDS and 0.5mL of 20mg/mL proteinase K at 55°C for 1hr. The DNA fragments were extracted by phenol chloroform, amplified using the provided barcode primers with 15 cycles of PCR. CUT&Tag libraries were cleaned with a single round of Novo NGS DNA clean beads (provided in the kit) at a 1.3 to 1 (vol/vol) ratio of beads to sample, quantified and pooled for sequencing. The DNA fragments were extracted by phenol chloroform, amplified using the provided barcode primers with 15 cycles of PCR. CUT&Tag libraries were cleaned with a single round of Novo NGS DNA clean beads (provided in the kit) at a 1.3 to 1 (vol/vol) ratio of beads to sample, quantified and pooled for sequencing.