title: GSM8006375: H3K18bu_CUTnTag_but1; Mus musculus; OTHER
library_name: GSM8006375
library_construction_protocol: CUT&Tag was performed using CUT&Tag-ITTM assay kits (Active Motif # 53160, 53170). ~500,000 viable nuclei (30 mg tissue) were used per reaction. Tissue samples were Dounced 20 times with a loose pestle and 10 times with a tight pestle. 5 μg of undiluted primary antibody was added to each sample for an overnight incubation at 4C with orbital mixing. 100 μL of 1:100 of Guinea Pig Anti-Rabbit secondary antibody was then added to each reaction and the samples were incubated on an orbital rotator for 60 min at RT. Assembled pA-Tn5 Transposomes were added to each sample and reactions were incubated on an orbital rotator for 60 min at RT. 125 μL of Tagmentation Buffer (5 mL Dig-300 buffer and 50 µL 1 M MgCl2) was added to each sample and reactions were incubated at 37C for 1 h. Nextera i5 and i7 indexing primers were used and final PCR reactions were purified using Zymo DNA Clean and Concentrator-5 Kit (cat # D4013). Final PCR reactions were purified using Zymo DNA Clean and Concentrator-5 Kit (cat # D4013) CUT&Tag-ITTM assay kits (Active Motif # 53160, 53170) and Hatice S. Kaya-Okur et al., (Nat Comm 2019)