Fig. 2: Overview of the archival chromatin assay workflow.

We prepare heavily fixed archival tissue nuclei for chromatin extraction through a stepwise process. This includes cryo-pulverisation for tissue fracturing, enzymatic digestion with pepsin to improve dissociation, Dounce homogenisation for fine tissue disruption, and prolonged sonication (Nuclei Extraction by SONication). The tissue can then be processed via: Formaldehyde Assisted Isolation of Regulatory Elements (FAIRE) treatment with further sonication to shear the chromatin followed by reservation of a fraction for input control and phenol:chloroform extraction of the FAIRE fraction or Micrococcal Nuclease (MNase) treatment of the nuclei with co-digestion with MNase and Exonuclease III. Isolated chromatin then undergoes RNase and proteinase K treatment before DNA fragments are purified using phenol:chloroform extraction and SPRI bead purification optimised for small fragment recovery. Sequencing libraries are prepared using an IDT xGEN cfDNA & FFPE DNA kit for paired-end sequencing. Sequencing reads are mapped using kalign without prior trimming. Alignments are de-duplicated using unique molecular identifiers (UMIs) and undergo GC-bias correction. Enrichment analyses are performed using the DANPOS3 dpeak function. Prior to downstream analysis, confirmation of the expected peak loss versus peak gain pattern can be performed. Created with BioRender.com, released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.