GSM5589376: ATAC-Seq lung_SM-A62E9; Homo sapiens; ATAC-seq
Nuclei were extracted from flash frozen tissue either by manual pulverization via mortar and pestle while submerged in liquid nitrogen, or homogenization in a gentleMACS M-tube (Miltenyi) on a gentleMACS Octo Dissociator (Miltenyi) using the “Protein_01_01” protocol in MACS buffer (5 mM CaCl2, 2 mM EDTA, 1X protease inhibitor (Roche, 05-892-970-001), 300 mM MgAc, 10 mM Tris-HCL pH 8, 0.6 mM DTT) and pelleted with a swinging bucket centrifuge (500 x g, 5 min, 4°C; 5920R, Eppendorf). Extracted nuclei were permeabilized either in 1 mL Nuclear Permeabilization Buffer (1X PBS, 5% Bovine Serum Albumin, 0.2% IGEPAL CA-630 (Sigma), 1 mM DTT, 1X Protease inhibitor), or 1 mL OMNI Buffer (10mM Tris-HCL (pH 7.5), 10mM NaCl, 3mM MgCl2, 0.1% Tween-20 (Sigma), 0.1% IGEPAL-CA630 (Sigma) and 0.01% Digitonin (Promega) in water). After resuspension in permeabilization buffer, nuclei were rotated at 4 ˚C for 5 minutes before being pelleted with a swinging bucket centrifuge (500 x g, 5 min, 4°C; 5920R, Eppendorf). After centrifugation, permeabilized nuclei were resuspended in 500 μL high salt tagmentation buffer (36.3 mM Tris-acetate (pH = 7.8), 72.6 mM potassium-acetate, 11 mM Mg-acetate, 17.6% DMF) and counted using a hemocytometer. Concentration was adjusted to 2,000 nuclei/9 μl, and 2,000 nuclei were dispensed into each well of a 96-well plate per sample (96 tagmentation wells/sample, samples were processed in batches of 2-4 samples). For tagmentation, 1 μL barcoded Tn5 transposomes were added using a BenchSmart™ 96 (Mettler Toledo), mixed five times, and incubated for 60 min at 37 °C with shaking (500 rpm). To inhibit the Tn5 reaction, 10 µL of 40 mM EDTA (final 20mM) were added to each well with a BenchSmart™ 96 (Mettler Toledo) and the plate was incubated at 37 °C for 15 min with shaking (500 rpm). Next, 20 µL of 2x sort buffer (2 % BSA, 2 mM EDTA in PBS) were added using a BenchSmart™ 96 (Mettler Toledo). All wells were combined into a separate FACS tube for each sample, and stained with Draq7 at 1:150 dilution (Cell Signaling). Using a SH800 (Sony), 20 nuclei per sample were sorted per well into eight 96-well plates (total of 768 wells) containing 10.5 µL EB (25 pmol primer i7, 25 pmol primer i5, 200 ng BSA (Sigma)). Preparation of sort plates and all downstream pipetting steps were performed on a Biomek i7 Automated Workstation (Beckman Coulter). After addition of 1 µL 0.2% SDS, samples were incubated at 55 °C for 7 min with shaking (500 rpm). 1 µL 12.5% Triton-X was added to each well to quench the SDS. Next, 12.5 µL NEBNext High-Fidelity 2× PCR Master Mix (NEB) were added and samples were PCR-amplified (72 °C 5 min, 98 °C 30 s, (98 °C 10 s, 63 °C 30 s, 72°C 60 s) × 12 cycles, held at 12 °C). After PCR, all wells were combined. Libraries were purified according to the MinElute PCR Purification Kit manual (Qiagen) using a vacuum manifold (QIAvac 24 plus, Qiagen) and size selection was performed with SPRISelect reagent (Beckmann Coulter, 0.55x and 1.5x). Libraries were purified one more time with SPRISelect reagent (Beckman Coulter, 1.5x). Libraries were quantified using a Qubit fluorimeter (Life technologies) and a nucleosomal pattern of fragment size distribution was verified using a Tapestation (High Sensitivity D1000, Agilent). Libraries were sequenced on a NextSeq500 or HiSeq4000 sequencer (Illumina) using custom sequencing primers with following read lengths: 50 + 10 + 12 + 50 (Read1 + Index1 + Index2 + Read2). OTHER: single cell ATAC-seq