GSM5224984: EL4 T-bet cells unstimulated; Mus musculus; ATAC-seq
50,000 cells were washed in 50 μl cold PBS (500g, 5 min, 4°C) and re-suspended in 50 μl cold lysis buffer I (10 mM Tris-Cl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5% IGEPAL CA-630) and spun (500g, 10 min, 4°C). The supernatant was removed, and lysed cells re-suspended in 50 μl transposase reaction mix, containing 10 mM Tris pH8.0, 5 mM MgCl2, 10% dimethylformamide and 2.5 μl Tn5 transposon + transposase reagent (TDE1 Illumina). Reactions were incubated at 37°C for 45 min, with shaking at 500 rpm. Following transposition, 150 μl reverse-crosslinking buffer (50 mM Tris-Cl, 1 mM EDTA, 1% SDS, 0.2 M NaCl) was added to reactions, and incubated at 65°C overnight at 1000 rpm before DNA purification, using the MinElute PCR purification kit (Qiagen). Transposed DNA was amplified by PCR for 5 cycles, using KAPA HiFi HotStart ReadyMix, 1.25 μM Ad1 primer and 1.25 μM Ad2.x indexed primers, as described (Buenrostro et al., 2013). 5 μl of each reaction was taken for qPCR as described, using KAPA HiFi HotStart ReadyMix and 50X KAPA Low ROX for signal normalisation, to determine the additional number of PCR cycles for library amplification. PCR reactions were purified using MinElute gel extraction kit (Qiagen) according to the manufacturer's instruction, and fragments between 100-1000 bp in size were selected for sequencing. DNA Fragmentation of the library was measured using the Bioanalyzer High Sensitivity DNA assay (Agilent), and library concentration quantified by qPCR analysis, using Illumina Library Quantification kit (KAPA). DNA from multiple libraries was pooled at equimolar concentrations to a final concentration of 4 nM.
GEO Accession
GSM5224984