GSM6454830: CUT&RUN_ETS1_Th1_WT_Rep1B_Rep1T; Mus musculus; OTHER
CUT&RUN was performed on in-vitro polarized Th1 cells using CUTANA™ ChIC/CUT&RUN Kit (EpiCypher, Cat#14-1048), using manufacturer's recommendation. Briefly, 4 × 105 cells live cells were sorted out and nuclei were extracted, washed, and allowed to adsorb onto activated ConA beads. Cells were then resuspended in recommended buffer, 0.5 mg of antibody was added, mixed well, and allowed to incubate at 4°C overnight on a nutator. Recommended antibodies were used, including anti-H3K27ac (Acetyl-Histone H3 (Lys27) (D5E4) XP® Rabbit mAb, CST, Cat #8173S), anti-ETS-1 (C-20, SantaCruz, Cat# sc-350X) and anti-CTCF (Millipore, Cat# 07-729), along with positive and negative controls. Subsequently, the reactions were washed with cell permeabilization buffer and incubated with pAG-MNase, and the DNA was isolated for the antibody-binding regions. Libraries were prepared according to instructions accompanying the NEBNext Ultra II Library Prep Kit for Illuina (Part# E7645S). Indexed libraries were validated for quality and size distribution using a TapeStation 2200 (Agilent). Paired end sequencing (38 bp+38 bp) was performed on a NextSeq 550 or 61bp+61bp on Novaseq 6000 (Illumina) following the manufacturer's protocol.