GSM5289638: GM12878; Homo sapiens; OTHER
Tissue samples were minced on dry ice and resuspended in NIB (10 mM HEPES-KOH (Fisher Scientific, BP310-500 and Sigma Aldrich 1050121000, respectively), pH 7.2, 10 mM NaCl, 3mM MgCl2 (Fisher Scientific AC223210010), 0.1 % (v/v) IGEPAL CA-630 (Sigma Aldrich I3021), 0.1 % (v/v) Tween (Sigma-Aldrich P-7949) and diluted in PCR-grade Ultrapure distilled water (Thermo Fisher Scientific 10977015)). After a 5 minute incubation in ice, tissue was dounce homogenized to isolate nuclei. For GM12878, cells were spun down and resuspended in NIB. See https://dx.doi.org/10.17504/protocols.io.bd6wi9fe and https://dx.doi.org/10.17504/protocols.io.beb3jaqn for more details. Tagmentation plates were prepared by the combination of 420 uL of 1400 nuclei/uL solution with 540 uL 2X TD Buffer (Nextera XT Kit, Illumina Inc. FC-131-1024). From this mixture, 8uL (~5000 nuclei total) was pipetted into each well of a 96 well plate dependent on well schema (Figure 2a). 1uL of 2.5uM uniquely indexed transposase was then pipetted into each well. Tagmentation was performed at 55°C for 10 minutes on a 300 rcf Eppendorf ThermoMixer. Following this incubation, plate temperature was brought down with a brief incubation on ice to stop the reaction. Dependent on experimental schema pools of tagmented nuclei were combined and 2uL 5mg/mL DAPI (Thermo Fisher Scientific D1306) was added.  Nuclei were then flow sorted via a Sony SH800 to remove debris and attain an accurate count per well prior to PCR. A receptacle 96 well plate was prepared with 9uL 1X TD buffer (Nextera XT Kit, Illumina Inc. FC-131-1024,diluted with ultrapure water), and held in a sample chamber kept at 4°C. Fluorescent nuclei were then flow sorted gating by size, internal complexity and DAPI fluorescence for single nuclei following the same gating strategy as previously described40. Immediately following sorting completion, the plate was sealed and spun down for 5 minutes at 500 rcf and 4°C to ensure nuclei were within the buffer.  Nucleosomes and remaining transposases were then denatured with the addition 1uL of 0.1% SDS (~0.01% f.c.) per well. 4uL of NPM (Nextera XT Kit, Illumina Inc) per well was subsequently added to perform gap-fill on tagmented genomic DNA, with an incubation at 72°C for 10 minutes. 1.5 uL of 1uM A14-LNA-ME oligo was then added to supply the template for adapter switching (Supplementary Table 3). The polymerase based adapter switching was then performed with the following conditions: initial denaturation at 98°C for 30 seconds, 10 cycles of 98°C for 10 seconds, 59°C for 20 seconds and 72°C for 10 seconds. The plate was then held at 10°C. After adapter switching 1% (v/v) Triton-X 100 in ultrapure H2O (Sigma 93426) was added to quench persisting SDS. At this point, some plates were stored at -20°C for several weeks while others were immediately processed.   The following was then combined per well for PCR: 16.5 ul sample, 2.5uL indexed i7 primer at 10 uM, 2.5uL indexed i5 primer at 10 uM, 3 uL of ultrapure H2O, and 25 uL of NEBNext Q5U 2X Master mix (New England Biolabs M0597S), and 0.5uL 100X SYBR Green I (Thermo Scientific S7563) for a 50 uL reaction per well (Supplementary Table 4-5). A real time PCR was performed on a BioRad CFX with the following conditions, measuring SYBR fluorescence every cycle: 98°C for 30 seconds; 16-18 cycles of 98°C for 10 seconds, 55°C for 20 seconds, 72°C for 30 seconds, fluorescent reading, 72°C for 10 seconds. After fluorescence passes an exponential growth and begins to inflect, the samples were held at 72°C for another 30 seconds then stored at 4°C.