title: GSM3576362: Fetal cells biological replicate 2; Mus musculus; ATAC-seq
library_construction_protocol: The normal mammary epithelial cells were dissected and FACS isolated as EpCAM+ and Lin- from primary mammary tissue (see manuscript for details). Cells were thawed and pelleted in a swinging bucket centrifuge (500 x g, 5 min, 4°C; 5920R, Eppendorf). Cell pellets were resuspended in 250 µl nuclei permeabilization buffer (10 mM Tris-HCl pH 7.4 (Sigma), 10 mM NaCl, 3 mM MgCl2, 0.1% GEPAL-CA630 (Sigma), 0.1% Tween-20, and 0.01% Digitonin (Promega, G9441), cOmplete (Roche) in water) (PMID: 28846090), incubated for 5 min at 4 °C with rotation and pelleted again (500 x g, 5 min, 4°C; 5920R, Eppendorf). 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 2000 nuclei/9 µl, and 2,000 nuclei were dispensed into each well of a 96-well plate - 32 wells for each of the adult replicates and 16 wells for the fetal replicates, respectively. All downstream pipetting steps were performed on a Biomek i7 Automated Workstation (Beckman Coulter). For tagmentation, 1 µL barcoded Tn5 transposomes (PMID: 25079858) was added, mixed and incubated for 60 min at 37 °C with shaking (500 rpm). To inhibit the Tn5 reaction, 10 µL of 40-mM EDTA were added to each well and the plate was incubated at 37 °C for 15 min with shaking (500 rpm). Next, 5 µL 5 x sort buffer (5 % BSA, 5 mM EDTA in PBS) were added. All wells were combined and filtered using a 30-µm CellTric (Sysmex) into a FACS tube and stained with 3 µM Draq7 (Cell Signaling). Using a SH800 (Sony), 20 nuclei 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)). After addition of 1 µL 0.2% SDS, samples were incubated at 55 °C for 7 min with shaking (500 rpm). We added 1 µL 12.5% Triton-X to each well to quench the SDS and 12.5 µL NEBNext High-Fidelity 2× PCR Master Mix (NEB). Samples were PCR-amplified for 12 cycles (72 °C 5 min, 98 °C 30 s, (98 °C 10 s, 63 °C 30 s, 72 °C 60 s) × 11, held at 72 °C). After PCR, all wells were combined. Libraries were purified according to the MinElute PCR Purification Kit manual (Qiagen) and size selection was performed with SPRI Beads (Beckmann Coulter, 0.55x and 1.5x). Libraries were quantified using a Qubit fluorimeter (Life technologies) and the nucleosomal pattern was verified using a Tapestation (High Sensitivity D1000, Agilent). snATAC-seq (PMID: 29434377, 25953818). Library for each sample was sequenced twice (v1 and v2) to obtain enpugh read depth.
attributes.tag: GEO Accession
attributes.value: GSM3576362