GSM4908679: NKT1 lung rep2; Mus musculus; ATAC-seq
Cells representing specific iNKT subsets were sorted directly into 0.2 ml PCR tubes containing 8 µL of low-input lysis buffer (0.2% Triton-X-100 and RNase inhibitor) and stored at -80oC until processed further.  RNA extraction, reverse transcription, cDNA amplification, fragmentation, and additional PCR amplification was performed as previously described (Rosales et al. Methods Mol Biol 1799, p275). iNKT cells (10,000) were sorted into 1.5 mL Eppendorf tubes containing PBS with 5% FCS.  Cells were centrifuged at 600g for 10 minutes at 4oC, washed with 50 mL PBS, then resuspended in 50 mL ATAC lysis buffer (10 mM Tris pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40).  Cells were centrifuged in lysis buffer for 10 minutes at 600g, 4oC.  Following lysis, the pellet was resuspended in 50 mL ATAC reaction mix (25uL 2X TD buffer, 2.5 mL Nextera Enzyme, 22.5 mL water, Illumina).  The transposase reaction was carried out at 37oC for 30 minutes. Libraries were amplified using a KAPA HiFi real-time library amplification kit with barcoded primers for 11-12 cycles followed by 2x50 cycle paired-end sequencing.