GSM2753114: mm_atac_pre-prob.2; Mus musculus; ATAC-seq
To prepare Nuclei, respective flow sorted mouse or EBV transformed human peripheral B cells (1x105) cultured in RPMI media (with L- glutamine, 10% FBS, 1mM Sodium pyruvate, 10mM HEPES, 100units/ml penicillin, 100µg/ml Streptomycin) were centrifuged at 500g for 5 min, which was followed by a wash with ice-cold PBS and centrifugation at 500g for 5 min. Cells were lysed using cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2and 0.1% IGEPAL CA-630). Immediately after lysis, nuclei were spun at 500g for 10 min at 40C and supernatant removed. The nuclei pellet was resuspended in the transposase reaction mix (25 μL 2X Tagment buffer, 2.5 μL Tagment DNA enzyme (Illumina, FC-121-1030) and 22.5 μL nuclease-free water). The transposition reaction was carried out at 37 °C for 30 min. Directly following transposition the sample was purified using a Qiagen MinElute kit. Following purification, we amplified library fragments using Nextera PCR Primers (Illumina Nextera Index kit) and NEBnext PCR master mix (New England lab, 0541) for a total of 10-12 cycles. The libraries were then purified using a Qiagen PCR cleanup kit.   The amplified, adapter ligated libraries were size selected using Life Technologies' E-Gel® SizeSelect™ gel system in the 150-650bp range. The size-selected libraries were quantified using the Agilent Bioanalyzer and via q-PCR in triplicate using the KAPA Library Quantification Kit on the Life Technologies Step One System. Libraries were sequenced on the Illumina Hiseq2500 system to generate 75-100M, 50bp paired end reads.