GSM8442322: RNA-seq on NPCs derived from WTC11-iPSCs (biol rep2); Homo sapiens; RNA-Seq
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. Total RNA from NPCs was extracted using Direct-Zol RNA Miniprep kit (Zymo Research, R2052). Samples were depleted of ribosomal RNA (rRNA) using the NEBNext rRNA Depletion kit v2 (NEB, E7400L). Libraries for sequencing were prepared using the NEBNext Ultra II directional library kit (NEB, E7760) following the manufacturer's protocol. Libraries were quantified by Tapestation D1000 (Agilent) and Qubit high sensitivity dsDNA (Invitrogen) assays, pooled, and sequenced on NovaSeq 6000 or NovaSeq X Plus systems (Illumina).