GAM methods and data processing
Preparation of cryosections
H1 cells were fixed and processed for cryosectioning as described previously140. In brief, H1 cells were grown to 70% confluency, the medium was removed and cells were fixed in 4% and 8% paraformaldehyde in 250 mM HEPES-NaOH (pH 7.6; 10 min and 2 h, respectively), gently scrapped and softly pelleted, before embedding (>2 h) in saturated 2.1 M sucrose in PBS and frozen in liquid nitrogen on copper sample holders. Frozen samples were stored in liquid nitrogen. Ultrathin cryosections were cut using a Leica ultracryomicrotome (UltraCut EM UC7, Leica Microsystems) at approximately 220 nm thickness, captured on sucrose-PBS drops and transferred to 4 µm PEN steel frame slide for laser microdissection (Leica Microsystems, 11600289). Sucrose embedding medium was removed by washing with 0.2-μm-filtered molecular biology grade PBS (3 × 5 min each) and filtered ultrapure water (5 min). For laser microdissection, cryosections on PEN membranes were washed, permeabilized and incubated (2 h, room temperature) in blocking solution (1% BSA (w/v), 5% FBS (w/v, Gibco 10270), 0.05% Triton X-100 (v/v) in PBS). After incubation (overnight, 4 °C) with primary anti-pan-histone (1:50) antibody (Merck, MAB3422) in blocking solution, the cryosections were washed three to five times for 30 min in 0.025% Triton X-100 in PBS (v/v) and immunolabelled (1 h, room temperature) with secondary antibodies in blocking solution, followed by three (15 min) washes in PBS.
Isolation of NPs
Nuclear staining was visualized using a Leica laser microdissection microscope (Leica Microsystems, LMD7000) using a ×63 dry objective. Individual nuclear profiles (NPs) were laser microdissected from the PEN membrane, and collected into PCR adhesive caps (AdhesiveStrip 8C opaque; Carl Zeiss Microscopy 415190-9161-000). GAM data were collected in multiplexGAM mode, where three NPs are collected into each adhesive cap. The presence of NPs in each lid was confirmed with a ×5 objective using a 420–480 nm emission filter. Control lids not containing NPs (water controls) were included for each dataset collection to keep track of contamination and noise amplification of whole genome amplification and library reactions. Collected NPs were kept at −20 °C until whole-genome amplification.
WGA
Whole genome amplification (WGA) was performed as described previously141 with minor modifications. In brief, DNA was extracted from NPs at 60 °C in the lysis buffer (20 mM Tris-HCl pH 8.0, 1.4 mM EDTA, 560 mM guanidinium-HCl, 3.5% Tween-20, 0.35% Triton X-100) containing 0.75 U ml−1 Qiagen protease (Qiagen, 19155). After 24 h of DNA extraction, the protease was heat-inactivated at 75 °C for 30 min and the extracted DNA was amplified by two rounds of PCR. First, quasi-linear amplification was performed with random hexamer GAT-7N primers with an adaptor sequence. The lysis buffer containing the extracted genomic DNA was mixed with 2× DeepVent mix buffer (2× Thermo polymerase buffer (10×), 400 µm dNTPs, 4 mM MgSO 4 in ultrapure DNase-free water), 0.5 µM GAT-7N primers (5′-GTGAGTGATGGTTGAGGTAGTGTGGAGNNNNNNN) and 2 U µl−1 DeepVent (exo-) DNA polymerase (New England Biolabs, M0259L) and incubated for 11 cycles in the BioRad thermocycler. The second exponential PCR amplification was performed in presence of 1x DeepVent mix, 10 mM dNTPs, 0.4 µM GAM-COM primers (5′-GTGAGTGATGGTTGAGGTAGTGTGGAG) and 2 U µl−1 DeepVent (exo-) DNA polymerase in the programmable thermal cycler for 26 cycles. WGA was performed in 96-well plates using Microlab STARLine liquid handling workstation (Hamilton).
Preparation of GAM libraries for high-throughput sequencing
After WGA, the samples were purified using the SPRI magnetic beads (1.7× ratio of beads per sample volume). The DNA concentration of each purified sample was measured using the Quant-iT PicoGreen dsDNA assay kit (Invitrogen, P7589). Sequencing libraries were then made using the in-house tagmentation-based protocol. After library preparation, the DNA concentration for each sample was measured using the Quant-iT PicoGreen dsDNA assay, and equal amounts of DNA from each sample was pooled together. The final pool of libraries was analysed using DNA High Sensitivity on-chip electrophoresis on an Agilent 2100 Bioanalyzer and sequenced on Illumina NextSeq 500 machine.
GAM data sequence alignment
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