title: GSM8650256: 166; Homo sapiens; ATAC-seq
library_construction_protocol: The research materials were samples of nuclei isolated from PBMC cell suspensions. Nuclei were isolated using the following method: cells were pelleted and resuspended in 100 μl of lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% Nonidet P40 Substitute, 0.01% Digitonin, 1% BSA, nuclease-free water), incubated for 5 min and added 1 ml of wash buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, nuclease-free water) , the nuclei were precipitated for 5 min at 500 rcf at a temperature of 4°C, then resuspended in the prepared Diluted Nuclei Buffer. Nuclear quality control was carried out using a Countess II FL counter and cell viability analyzer and an EVOS M7000 microscope. For sample preparation of libraries, nuclei were used that met the requirements of the protocol. The sample of nuclei under study was added to the prepared Transposition Mix, 10 μl per sample. The volume of the sample was calculated based on the concentration of nuclei using the formula (10000*1.53)/n = x, where n is the concentration of nuclei in the sample, x is the required volume of the sample. The mixture was incubated for 60 min at 37°C. Next, 60 μl of the prepared Master Mix was added to the samples, mixed using an automatic dispenser 10 times and applied to the chip to generate an emulsion. Afterwards, gel particles and oil were applied to the chip. The loaded chip was processed at The Chromium Controller station. The resulting emulsion was amplified. After amplification, the emulsion was purified: 125 μl of Recovery Agent was added to each sample, the tubes with the mixture were carefully inverted 10 times, then briefly centrifuged and 130 μl of the lower pink phase was collected; This was followed by cleaning with magnetic Dynabeads MyOne SILANE and then cleaning with Beckman Coulter SPRIselect Reagent magnetic particles. After purifications, indexing PCR was performed. For it, we used Single Index Kit N Set A for one-way indexing. To 40 μl of sample, 57.5 μl of the prepared Sample Index PCR Mix and 2.5 μl of index were added. The number of cycles in the indexing PCR was set to 9. After indexing, the samples were subjected to double purification on magnetic particles and the quality of the resulting libraries was controlled using a Tape Station 4200 with a High Sensitivity D1000 kit, an Agilent Bioanalyzer 2100 with a High Sensitivity DNA chip kit, and a Qubit 4 fluorimeter. Ready-made libraries sequenced to a coverage of >250 million reads per sample.