GSM6509633: Pachynema ATAC-seq rep 1; Mus musculus; ATAC-seq
In order to prepare nuclei, 50,000 cells isolated by FACS were spun at 500g for 5 minutes immediately after sorting, followed by a wash using 50µl of cold 1 x PBS and centrifugation at 500g for 5 minutes. Cells were then lysed using a cold lysis buffer (10mM Tris-HCl, pH 7.4, 10mM NaCl, 3mM MgCl2 and 0.1% IPEGAL CA-630). Immediately following lysis, nuclei were spun at 500g for 10 minutes in a refrigerated centrifuge. In order to avoid cell loss during nuclei preparation, a fixed-angle centrifuge was used and cells were carefully pipetted away from the pellet after centrifugation. Nuclei were stored at -80°C. Stored nuclei were thawed on ice and the pellet was resuspended in a transposase reaction mixture (25µl 2 x TD buffer, 2.5µl transposase (Illumina) and 22.5µl nuclease-free water). The transposition reaction mixture was carried out for 30 minutes at 37˚C. Immediately following transposition, the sample was purified by means of a Qiagen MinElute kit. After purification, the library of fragments was amplified using 1 x NEBnext PCR master mix and 1.25µM of custom Nextera PCR primers 1 and 2 (Supplementary Table x).  The following PCR conditions were utilized: 72˚C for 5 minutes; 98˚C for 30 seconds; and thermocycling at 98˚C for 10 seconds, 63˚C for 30 seconds, and 72˚C for 1 minute. In order to reduce GC and size bias in the PCR analysis, PCR reactions were monitored using qPCR in order to stop amplification prior to saturation. This was done by amplification of the full libraries for five cycles, after which an aliquot of the PCR reaction was taken and 10µl of the PCR cocktail containing Sybr Green at a final concentration of 0.6x was added. This reaction was run for 20 cycles in order to determine the additional necessary number of cycles for the remaining 45µl reaction. The libraries were purified by means of a Qiagen PCR cleanup kit that yielded a final library concentration of ~30nM in 20µl. These libraries were amplified for a total of 10-12 cycles in order to generate the ATAC-seq libraries, which were prepared using the manufacturer's protocol (Illumina) and paired-end sequenced by means of Illumina NextSeq500 at Cornell University Biotechnology Resource Center.