GSM8390326: RPE-1, DSMO control,  Lamin AC rep3; Homo sapiens; OTHER
We perform Cleavage under targets and tagging (CUT&Tag) using Hieff NGS® G-Type In-Situ DNA Binding Profiling Library Prep Kit (Yeasen, Cat#12598ES48) against lamin A/C and lamin B1 following to the manufacture' s instruction. Briefly, for each sample, 105 cells were harvested after treatment with/without 0.4 μM APH for 48 hour and washed three times by PBS, resuspend  the cells with cell wash buffer gently, and incubate  with the concanavalin A coated magnetic beads previously washed by ConA bind buffer. Then concanavalin A beads coupled cells were collected and incubated with 2 μg primary antibody (mouse anti lamin A/C (Santa Cruz, Cat#sc-7292) or rabbit anti-lamin B1 (Abcam, Cat#ab16048)) and rabbit anti IgG (Thermo ) at 4 °C overnight with slow rotation. After removing primary antibodies using a magnetic stand, the secondary antibodies (goat anti-rabbit IgG (H+L) (Yeasen, Cat#34850ES60) and goat anti-mouse IgG (H+L) (Yeasen, Cat#3485ES60) were incubated with the concanavalin A beads coupled cells at room temperature for 1 h. Cells were washed three times with wash buffer to remove unbound antibodies. Then, the cells were incubated with pA/G-Transposome mix for 1 h at room temperature. To terminate tagmentation, terminate solution and proteinase K were added to the sample and incubated at 55℃ for 30 min. DNA was purified using DNA selection beads and elutes  in 21 μL ddH2O. For library amplification, 8 µL of DNA was mixed with a uniquely barcoded i5 and a uniquely barcoded i7 primer, and amplified using 2× Ultima Amplification Mix (Yeasen). The library was then purified using DNA selection beads and washed twice with 80% ethanol and eluted in 21 μL of ddH2O. Libraries were sequenced by Berry Genomic (Beijing, China) on an Illumina NovaSeq platform for 10 G per sample and 150-bp paired-end reads were generated.