GSM5530602: CHLA10_shNS_H3K4me3_1; Homo sapiens; OTHER
Cells were bound to concanavalin A-coated magnetic beads , permeabilized with digitonin, and bound with primary antibodies. Samples are then arrayed in a 96-well plate and processed on a Beckman Biomek FX liquid handling robot equipped with a 96S Super Magnet Plate. The MNase digestion reaction is performed at 0–4 °C after the addition of 2 mM CaCl2.  Chromatin fragments released into the supernatant arae collected for the next steps. Methods from (Janssens et al. 2018 Methodology: "Chromtin fragments are used as the substrate for end-repair and ligation with barcoded Y-adapters. Prior to ligation, the A-tailing step is performed at 58 °C to preserve sub-nucleosomal fragments in the library. End-repair and adapter ligation reactions were performed on a separate thermocycler. Chromatin proteins are then digested with Proteinase K, and adapter ligated DNA fragments are purified on the Biomek FX using two rounds of pre-PCR Ampure bead cleanups with size selection. PCR enrichment reactions are performed on a thermocycler using the KAPA PCR kit (KAPA Cat#KK2502). Two rounds post-PCR Ampure bead cleanups with size selection are performed on the Biomek FX to remove unwanted proteins and self-ligateed adapters. The size distributions of AutoCUT&RUN libraries were analyzed on an Agilent 4200 TapeStation, and library yield was quantified by Qubit Fluorometer (Thermo Fisher). Up to 24 barcoded AutoCUT&RUN libraries were pooled per lane at equimolar concentration for paired-end 25 × 25 bp sequencing on a 2-lane flow cell on the Illumina HiSeq 2500 platform at the Fred Hutchinson Cancer Research Center Genomics Shared Resource."