title: GSM8281594: Adult_Thymus_Double_Positive_ATAC_4; Mus musculus; ATAC-seq
library_name: GSM8281594
library_construction_protocol: Thymi and spleens from adult or neonatal mice were collected in RPMI+10% FBS and mashed through a 40µM cell strainer to create a single-cell suspension. Spleens and thymi underwent magnetic enrichment with CD8a and CD4 MicroBeads (Miltenyi Biotec; cat# 130-117-044; cat# 130-117-043), respectively, per the manufacturer's instructions. The bound thymic fraction was sorted for double positive thymocytes, while the eluted fraction underwent CD8a magnetic enrichment as described previously, after which the bound fraction was sorted for SP8 thymocytes, while the eluted fraction was sorted for DN thymocytes. Thymocyte counts were taken by diluting a single-cell thymus suspension and counting on a MoxiZ cell counter. The ATAC-seq libraries were prepared based on the Omni-ATAC-seq protocols  with some minor modifications. Briefly, 100,000 FACS-sorted cells were used to prepare nuclei. The transposition reaction was performed by incubating 25,000 nuclei with Nextera Tn5 transposase (Illumina) at 37°C for 1 hour. The transposition mixture was purified using a Zymo DNA Clean and Concentrator kit. The ATAC libraries were amplified for 11 cycles using NEBNext 2X MasterMix and Nextera Index primers. The amplified libraries were size-selected using AMPure beads, to remove large DNA fragments.