GSM4977279: In vitro 48h Th2 Rep 2; Mus musculus; ATAC-seq
Samples were prepared according to (Buenrostro et al., 2013). Briefly, 50,000 sort-purified Th17 cells were pelleted in a fixed rotor centrifuge at 500xg for 5 minutes, washed once with 50 μL of cold 1x PBS buffer. Spun down again at 500xg for 5 min. Cells were gently pipetted to resuspend the cell pellet in 50 μL of cold lysis buffer (10 mM Tris-HCl, pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) for 10 minutes. Cells were then spun down immediately at 500xg for 10 min and 4 degrees after which the supernatant was discarded and proceeded immediately to the Tn5 transposition reaction. Gently pipette to resuspend nuclei in the transposition reaction mix. Incubate the transposition reaction at 37 degrees for 30 min. Immediately following transposition, purify using a Qiagen MinElute Kit. Elute transposed DNA in 10 μL Elution Buffer (10mM Tris buffer, pH 8). The transposed nuclei were then amplified using NEBNext High-fidelity 2X PCR master mix for 5 cycles. In order to reduce GC and size bias in PCR, the PCR reaction is monitored using qPCR to stop amplification prior to saturation using a qPCR side reaction. The additional number of cycles needed for the remaining 45 μL PCR reaction is determined as following: (1) Plot linear Rn vs. Cycle (2) Set 5000 RF threshold (3) Calculate the # of cycle that is corresponded to ¼ of maximum fluorescent intensity. Purify amplified library using Qiagen PCR Cleanup Kit. Elute the purified library in 20 μL Elution Buffer (10mM Tris Buffer, pH 8).