title: GSM5098653: Input_Bcells; Homo sapiens; ChIP-Seq
library_construction_protocol: ChIP was performed as previously described [Langlais et al., JEM, 2016] with minor modifications. Briefly, cells were fixed by incubation with 1% formaldehyde in the culture medium for 10 minutes at room temperature. Crosslinking was stopped by adding 0.125 M glycine, cells were washed with cold PBS and nuclei were extracted. Nuclei were resuspended in sonication buffer and the chromatin was sheared during four 30 s sonication cycles separated by intervals of 30 s, with a Bioruptor Pico (Diagenode); the sonicated fragments were 100-500 bp in size. Bead immunocomplexes were prepared by overnight incubation of 20 μl of Dynabeads Protein G and 20 μl of Dynabeads Protein A (Life Technologies) with 5 μg of anti-c-Rel antibody (D5G1A) or with a rabbit normal IgG control (2729S), both from Cell Signaling Technologies. Immunoprecipitation was performed by overnight incubation of the antibody-bead matrices with sheared chromatin from the equivalent of 2.5x10^6 cells, followed by successive medium-stringency washes. Samples were de-crosslinked by overnight incubation at 65̊°C in 1% SDS buffer, followed by RNase A and proteinase K enzymatic treatments and were then subjected to DNA purification with the QIAquick PCR Purification kit (Qiagen). ChIP-seq libraries were prepared with the KAPA Hyperprep ChIP Library Preparation kit (Roche) and sequenced on one lane of an SP flow cell on an Illumina NovaSeq 6000 sequencer in a 50 bp paired-end read configuration. Input DNA for each cell type was sequenced as a negative control.