title: GSM4058205: Day10-CD8-N-CD19-1; Homo sapiens; ATAC-seq
library_construction_protocol: 100,000 cells from each sample were sorted by FACS into culture media, centrifuged at 500g at 4°C, then resuspended in ATAC-seq Resuspension Buffer (RSB) (10 mM Tris-HCl, 10 mM NaCl, 3mM MgCl2) supplemented with 0.1% NP-40,0.1% Tween-20, and 0.01% Digitonin.  Samples were split into two replicates each prior to all subsequent steps.  Samples were incubated on ice for 3 minutes, then washed out with 1 mL RSB supplemented with 0.1% Tween-20.  Nuclei were pelleted at 500g for 10 minutes at 4°C. The nuclei pellet was resuspended in 50 μL transposition mix (25 μl 2x TD buffer, 2.5 μl transposase (Illumina), 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, 5 μl H2O) and incubated at 37°C for 30 minutes in a thermomixer with 1000 RPM shaking.  The reaction was cleaned up using the Qiagen MinElute PCR Purification Kit. Libraries were PCR-amplified using the NEBNext Hi-Fidelity PCR Master Mix and custom primers (IDT) as described previously20. Libraries were sufficiently amplified following 5 cycles of PCR, as indicated by qPCR fluorescence curves20.  Libraries were purified with the Qiagen MinElute PCR Purification Kit and quantified with the KAPA Library Quantification Kit. Libraries were sequenced on the Illumina NextSeq at the Stanford Functional Genomics Facility with paired-end 75bp reads.
attributes.tag: GEO Accession
attributes.value: GSM4058205