GSM8647400: R6_H3K9ac_NeuN+_Rep3; Mus musculus; OTHER
NeuN+ and NeuN- sorted nuclei preparations from R6/1 and WT mice were divided to allow immunoprecipitation of the same extract with H3K9ac (Ab4441, Abcam), H3K18ac (C15410139, Diagenode) and IgG control (C15410206, Diagenode) with 50 000 nuclei/CUT&Tag. CUT&Tag was essentially performed as described by Kaya-Okur et al. (2020). Briefly, concanavalinA-coated beads (93569S, Cell signaling) were added to nuclei suspension at room temperature under rotation to attach nuclei. Then corresponding primary antibodies were incubated overnight at 4 °C. Beads-nuclei complex were washed and incubated 1h at RT with proteinA-Transposase5 complex (C01070001, Diagenode) to a final concentration of 1:250. Tagmentation was done by incubating samples 1h in MgCl2 at 37°C, then proteins were eliminated by 1h incubation in SDS and Proteinase K at 55°C. DNA was recovered using Qiagen MinElute PCR Purification Kit. CUT&Tag libraries were prepared from the eluted double-strand DNA using NEBNext HiFi 2x PCR master mix (BioLabs) and Illuminas compatible indexes (20091654, Illumina Nextera) (13 cycles). Amplified libraries were purified and size-selected using SPRI select beads (B23317, Beckmann Coulter) at 1.3 time, two times consecutively.