GSM4720311: human WBC DNase [GB930892_S51]; Homo sapiens; DNase-Hypersensitivity
DNase-seq human 1.25M 2-step crosslink human healthy T/B/NK/monocyte, final 0.1U/μl DNase digestion 37°C 5 minutes, T cell split #1-15 wells, B cell split #16-30 wells, NK cell split #31-45 wells, monocyte split #46-61 wells, TdT&T4 methods adding P7 barcode adaptor (12.5μl volumn/well 37°C 60 minutes), pool, dilution, counting and redistribute into 96-well plate, total 20 nuclei per well seal completely, 65°C overnight; direct PCR1 (12 cycles), pool all the DNA, EXO I 30 minutes, A-tailing and P5 ligation(rt overnight) , column purification and PCR2(16 cycles), run-e-gel and cut from 200-500bp rc#1 i5-01 The isolated 50M of PBMC suspended by 50ml PBS /MgCl2 are firstly fixed by adding 400μl freshly prepared 0.25M Disuccinimidyl glutarate (DSG, ThermoFisher Scientific, catalog no.20593) and incubate at room temperature for 45 minutes with rotation. After protein-protein fixation by DSG and washing three times by PBS, cells suspended by culture medium DMEM supplemented with 10% FBS are further fixed by the addition of 1:15 volume of 16% (w/v) methanol-free formaldehyde solution (Thermo Fisher Scientific) and incubate at room temperature for 10 minutes. The fixing reaction is terminated by a 1:10 volume of 1.25 M glycine and incubate at room temperature for 5 minutes. The fixed cells are collected by centrifugation at 1320rpm for 7 minutes and washed with PBS. The cells are stored in aliquots (1 × 106 cells per tube) at −80°C until use.
GEO
GSM4720311