GSM8442292: LaminB1 GO-CaRT on ESC-derived NPCs treated with DMSO for one week (biol rep1); Homo sapiens; OTHER
NPCs and neurons were harvested using Accutase. The cell pellet was resuspended in 1 mL ice cold PBS (Gibco). The cells were centrifuged at 500g for 3 min at 4 °C and the cell pellet was resuspended in 1 ml of nuclei isolation buffer (NIB: 10 mM HEPES-KOH, pH 7.9, 10 mM KCl, 0.1% NP40, 0.5 mM spermidine and 1× Halt protease inhibitor cocktail) and incubated for 10 min on ice. Nuclear pellet was collected by centrifuging at 600g for 3 min at 4°C and again resuspended in 1 ml of NIB to wash. Nuclei were fixed in 0.1% formaldehyde for 2 minutes and washed in NIB. Fixed nuclei were bound to concanavalin A beads  and ~50,000 nuclei were subjected to GO-CaRT/CUT&RUN and CUT&Tag. For GO-CaRT.ChIP-reChIP, LaminB1 GO-CaRT was performed on native (unfixed) nuclei and the LaminB1-enriched chromatin from the supernatant was used for subsequent sequential (2-step) ChIP. For Fab blocking experiments, HEK293T cells were harvested by trypsinization. Cells were fixed in 0.1% formaldehyde for 2 minutes at room temperature, followed by quenching in 0.1M Glycine (prepared in wash buffer). Cells were resuspended in 1 ml wash buffer (20 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 0.1% BSA, 0.5 mM spermidine (sigma) and 1× Halt protease inhibitor cocktail)) and centrifuged at 300g for 5 min. This step was repeated for a total of two washes. Cells were then bound to concanavalin A beads  and ~100,000 cells were subjected to CUT&RUN/GO-CaRT with or without Fab fragments. For all experiments, DNA was extracted using Phenol: Chloroform extraction method. Purified DNA fragments were analyzed by Tapestation High Sensitivity D1000 (Agilent), before proceeding to sequencing library construction. Sequencing libraries for GO-CaRT, CUT&RUN and LaminB1-GO-CaRT.ChIP-reChIP were generated using KAPA HyperPrep Kit (KAPA, KK8504) following the manufacturer's instructions. The libraries were amplified for 12–14 PCR cycles. DNA fragments were quantified by Tapestation D1000 (Agilent) and Qubit high sensitivity dsDNA (Invitrogen) assays, pooled, and sequenced by 150-bp paired-end sequencing on NovaSeq 6000 or NovaSeq X Plus systems (Illumina). CUT&RUN, CUT&Tag, GO-CaRT and  GO-CaRT followed by sequential ChIP (GO-CaRT.ChIP-reChIP).