GSM6203126: siRNA_1_CUTnTAG; Homo sapiens; OTHER
Cleavage Under Targets and Tagmentation (CUT&Tag) is a method to investigate genomic localization of histone modifications and some transcription factors to reveal interactions between proteins and DNA or identifying DNA binding sites for proteins of interest. CUT&Tag is based on the same principles as ChIP-Seq but with several changes to the protocol, where fresh cells are bound to concanavalin A beads and the antibody incubation is performed with cells in their native state. Directly following antibody binding, the chromatin is sheared, tagmentation performed using the protein A-Tn5 (pA-Tn5) transposome and NGS libraries are prepared. CUT&Tag-IT™ Assay Kits (Catalog No. 53160Active Motif North America) were used for tagmentation and library preparation as per kit procedures. Briefly, 50,000 to 100,000 cells were harvested and washed with 1X wash Buffer supplemented with Protease Inhibitor Cocktail. Then the cells were bound to Concanavalin A beads and incubated with 1ry antibody overnight at 4°C with orbital mixing. This was followed by 2 hours incubation with Guinea Pig Anti-Rabbit Antibody secondary antibody 1:100 in Dig-Wash Buffer. Cells were incubated for 60 minutes with pA-Tn5 Transposomes diluted to 1:100 in Dig-300 buffer. Then 50 μl of Tagmentation Master Mix was added to each sample and incubated at 37°C for 30 minutes in a thermomixer set at 800 rpm. This was followed by extraction and amplification of tagmented DNA with library preparation using unique combinations of i7/i5 primers which are based on Illumina's Nextera adapters. Finally, the library was purified by SPRI bead clean-up.