title: GSM7901716: Eomes-KO CD4 T cells (replicat 2) [CnR]; Mus musculus; OTHER
library_name: GSM7901716
library_construction_protocol: CUT&RUN was performed as previously described (Krzywinska et al., 2022) with some modifications. Briefly, 50,000 cells were harvested and processed with the CUT&RUN (Cleavage Under Targets & Release Using Nuclease) Assay Kit (86652; Cell Signaling Technology) following the supplier's instructions. Eomes monoclonal antibody (Dan11mag, Thermofisher, 14-4875-82) was used at 2 µg. DNA was purified using the QIAGEN MinElute kit (QIAGEN) and its quality was assessed on a 2100 Bioanalyzer (Agilent Technologies). Paired-end libraries have been prepared according to Active Motif's protocol with some adjustments, using the Next Gen DNA Library Kit (Active Motif, Carlsbad, USA), assuming a starting input of 100 pg of dsDNA. Briefly, DNA first underwent a 2-phases reparation. Then, Illumina compatible adaptors were ligated, allowing the barcoding of the samples with single indices, and the addition of molecular identifiers (MIDs) composed of 9-bases random sequence. These MIDs will enable to distinguish PCR duplicates from fragmentation duplicates, and the removal of true PCR duplicates from the subsequent sequencing analysis. The libraries were then amplified using 15 cycles of PCR, and an additional 2-sides purification step was finally performed, allowing to obtain 250-1000 pb fragments.