GSM9848466: 42077_SEM-IAA-50U-10min_2-1; Homo sapiens; OTHER
Cells were poured into methanol-free formaldehyde (final 1% concentration) at room temperature with gentle mixing for 10 min. Reactions were quenched with 3M Tris (pH 8.0), with 2 moles of Tris per 1 mole of formaldehyde, for at least 5 min. Fixed cells were washed with PBS, lysed with detergent containing buffers and needle lysis. Chromatin was digested with benzonase. Benzonase-fragmented chromatin was centrifuged at 16,363 x g for 15 min at 4˚C. The supernatant and pellet were separately used for library preparation. The pellet was resuspended by needle lysis with increasing needle gauge to minimize clumps. In some cases, total, super, or pellet fractions underwent DNA purification before being used for library input. Depending on the preparation, typically 0.5-1.3 million cell-equivalents of Benzonase-digested chromatin was digested with ProteinaseK for approximately 16 hours. Reverse-crosslinked DNA was purified with Ampure beads at 1.8x ratio to starting material and eluted in pure water. DNA underwent end-repair, A-tailing, and ligation to fork adapters (annealed Multiplexing Read 2 Sequencing primer /Nextera Read 2 External Adapter) with 3' Ts. Components for enzymatic reactions were added successively to the previous resulting DNA and the enzyme was then heat inactivated before adding the next master mix. The ligated DNA was purified again with Ampure beads at 1.8x ratio to the final volume of ligation reaction and then amplified by PCR using ExB2 and ExC1 primers. Amplified libraries were size selected by gel electrophoresis in the range from 200-500 bp. Libraries were sequenced with the Illumina NextSeq 550, paired-end mode. Illumina or Element library prep