GSM2627276: CD4_fixed_cell_ATAC_seq_rep; Homo sapiens; ATAC-seq
A total of 100k human CD4+ T resting cells, fixed or native (not fixed) were washed with PBS.  The cell pellets were washed with RSB buffer (10mM Tris-HCl, pH 7.5, 10mM NaCl, 3mM MgCl2) once and resuspended in 52 μl  cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.5% IGEPAL CA-630) and incubated on ice for 15min. add 6 ul 10x reaction buffer and 2ul ATAC-seq Tnp complex. Put in PCR machine for 37 °C 30 min. Add  3 μl 0.5M EDTA to stop the reaction. Add 40 μl H2O + 3 μl 10% SDS +5 μl proteinase K. put the samples in a 65 °C incubator overnight. Transfer samples to a new Eppendorf and extract DNA by phenol-chloroform extraction and ethanol precipitation. The libraries were processed same as Hi-C protocol (Lieberman-Aiden et al., 2009, Science. 2009 Oct 9;326(5950):289-93) The ATAC-seq Tnp comple-tagmented DNA pellets were resuspended in 10 μl of elution buffer (EB). Set up PCR by adding 25 μl of master Phusion HF mix, 22 μl H2O, 1 μl of one of the 10uM N501-N508 and 1 μl of one of the 10uM N701-N712. PCR: 72°C for 5min, 98°C for 45 sec. 19 cycles of ( 98°C, 15”; 65°C, 30”; 72°C, 30”), 72°C 5min. 4°C hold. Gel purify the 200-500 bp smear/fragments for sequencing.