Instrument: NextSeq 550
Strategy: ATAC-seq
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: Cells were purified by FACS-sorting. chromatin were digested by tn5 transposase at 37°C for 20 minutes.DNA fragments were extracted using MiniElute columns (Qiagen), and then amplified by PCR. FACS-sorted cells were centrifuged at 500 g for 20 m at 4 °C, and the pellet was suspended with 20 μL of lysis buffer (10 mM Tris HCl (pH 7.4), 5 mM MgCl2, 10% DMF, 0.2% N-P40) by pipetting 6-10 times to release the nuclei without purification. The cell lysate was then mixed with 30 μL reaction buffer (10 mM Tris HCl (pH 7.4), 5 mM MgCl2, 10% DMF, and homemade Tn5 Transposase) by vortexing for 5 s. The reaction was incubated at 37°C for 20 minutes, followed by DNA purification using a Qiagen MinElute kit. Libraries were amplified by PCR with indexed primers DNA fragments are amplified by PCR for 5 cycles using NEBnext master mix, with primers containing various indexes. Additional cycles are determined by qPCR on 1/10 of the amplified libraries. After amplification, the libraries are cleaned using AMPURE beads, to only keep the fragments larger than 100bp. The libraries are pooled before sequencing.