GSM9104253: 2nd replicate of H3K4me1 CUT&RUN on SALE-RIT1 M90I cells.; Homo sapiens; OTHER
12 million cells per cell line were collected by trypsinization, washed extensively in Wash Buffer (20mM HEPES-NaOH pH 7.5, 150mM NaCl, 0.5mM Spermidine (Cell Signaling Technology), and 1 large Roche complete EDTA-free protease tablet per 50mL solution). Concanavalin A beads (Bangs Laboratories, ca. no. BP531) were activated through repeated washing in Binding Buffer (20mM HEPES-KOS pH 7.9, 10mM KCl, 1mM CaCl2, 1mM MnCl2), and prepared at 10µL per sample. Beads were added under gentle vortex, and incubated rocking at room temperature for 8 minutes. Bead-cell complexes were collected by magnet separation, buffer removed, and bead-cell complexes were resuspended in Antibody Buffer (0.1% Triton-X 100, 2mM EDTA). Each cell line sample was then split into two technical replicates per cell line, per antibody. Primary antibodies were added to a final dilution of 1:100 (see Star Methods) and incubated for 2hrs rotating at 4 ºC. Samples were processed for AutoCUT&RUN by arraying in a 96-well PCR plate and processed on a Beckman Biomek i7 liquid handling instrument equipped with a 96S Super Magnet Plate (Alpaqua SKU A001322) for magnetic separation of samples during wash steps, and a QInstruments ColdPlate heater-cooler thermo block (QInstruments no. 2016-0110) with a 96 well PCR plate adapter (Qinstruments no. 2016-1041) to perform the MNase digestion reaction at 4 °C after the addition of 2 mM CaCl2. MNase digestion reactions are then stopped after 120 min by adding EGTA, which allows Mg2+ addition for subsequent enzymatic reactions. Chromatin fragments released into the supernatant during digestion are then 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 are performed on a separate thermocycler. Chromatin proteins are then digested with Proteinase K, and adapter ligated DNA fragments are purified on the Biomek i7 using two rounds of pre-PCR Ampure bead cleanup with size selection. PCR enrichment reactions are performed on a thermocycler using the KAPA PCR kit (KAPA Cat#KK2502). Two rounds of post-PCR Ampure bead cleanup with size selection are performed on the Biomek i7 to remove unwanted proteins and self-ligated adapters. The size distributions of AutoCUT&RUN libraries are analyzed on an Agilent 4200 TapeStation, and gated size and quantification values are used to calculate sample library yields for pooling. Up to 96 barcoded AutoCUT&RUN libraries are pooled at equimolar concentration for paired-end 50 × 50 bp sequencing on an Illumina NextSeq P2 platform at the Fred Hutchinson Cancer Center Genomics Shared Resource.