GSM8878937: rep2, mESCs, input; Mus musculus; ChIP-Seq
ChIP sequencing was performed by the Deep Sequencing unit of the MPI-IE using the RELACS nuclei barcoding protocol. WT26 mESCs were fixed by double cross-linking (2 mM DSG and 1% formaldehyde), snap-frozen in liquid nitrogen and stored at -150°C. Digested chromatin inside nuclei is end-repaired and A-tailed using end-repair and A-tailing components from NEBNext Ultra II DNA library preparation kit (E7645L, NEB) with the following modifications: 1.5 µL of end prep enzyme mix and 3.5 µL of reaction buffer are added to each 25 µL digested nuclei aliquot. Samples are mixed and incubated at 20 °C for 30 min followed by heat inactivation at 65 °C for 5 min. 1.2 µL of hairpin adapter nuclear barcodes is added to each sample. Ligation of barcodes to A-tailed chromatin is performed using components from NEBNext Ultra II DNA library preparation kit, with the following modifications: per sample 15 µL of ligation master mix and 0.5 µL of ligation enhancer is added, followed by 15 min incubation at 30 °C and 15 min incubation at 20 °C. Libraries with insert of 200-1000 bp were generated. The average library size is approximately 300-400 bp. Libraries were sequenced paired-end with a read length of 100 bp on the Illumina NovaSeq 6000 instrument.