GRHL and PGR control WNT4 expression in the mammary gland via 3D looping of conserved and species-specific enhancers [ATAC-Seq]
This dataset belongs to a study in which we functionally dissect the cis-acting enhancer network and spatiotemporal transcriptional mechanisms that regulate WNT4 expression in the mouse mammary gland and the human breast. As part of these efforts, we characterized the chromatin accessibility landscape in the pubertal mouse mammary gland. For this, we FACS sorted basal, luminal and stromal cell populations and performed bulk ATAC-seq analysis Overall design: The 3rd and 4th mammary gland fat pads from 6 puberty (p35) FVB/N mice were dissected, chopped to small pieces and incubated for 2h at 37°C in an orbital shaker in a digestion mix composed of DMEM/F12, 5 % FBS, 1% Penicillin / Streptomycin, 25mM HEPES and 300 U/ml Collagenase IV. Red blood cells were lysed with ACK Lysing Buffer and single cell suspensions were generated by consecutive digestion steps with Trypsin-EDTA and DNAseI. Cells were stained in HBSS containing 10% FBS with the following antibodies (eBioscience): anti-Mouse CD45-Biotin (clone 30-F11), anti-Mouse CD31-Biotin (clone 390), anti-Mouse TER-119-Biotin (clone Ter-119), anti-Mouse CD326-PE (clone G8.8), anti-Mouse CD49f-FITC (clone GoH3) and Streptavidin-APC. DAPI was used for live/dead cell discrimination. 55.000 luminal, basal and stromal cells were sorted into ice-cold 10mM HEPES containing 10% FBS using a BD FACSAria III equipped with a 100 um nozzle at 20psi. ATAC-seq samples were prepared according to the protocol from Buenrostro et al. (2013, 2015). Briefly, freshly sorted cells were washed once in 50ul ice-cold PBS and gently resuspended in 50ul cold lysis buffer (10mM Tris-HCl, pH 7,4; 10mM NaCl; 3mM MgCl2 and 0.1% NP-40) to isolate nuclei. The transposase reaction (Illumina Nextera DNA Library Preparation Kit, FC-121-1030) was performed for 30 min at 30°C and DNA was purified using MinElute PCR purification columns (Qiagen, #28004). DNA fragments were amplified using custom primers (Buenrostro et al., see table below) and NEBNext Ultra II Q5 Master Mix (#M05445). The following PCR conditions were used: 72 °C for 5 min; 98°C for 30 sec, 8 cycles of 98°C for 10 sec, 63°C for 30 sec and 72 °C for 1 min. Libraries were purified with the MinElute PCR purification kit. An additional, single left-sided purification with AMPure XP beads (Beckman Coulter #A63880) was performed to remove primer dimers. Libraries were quantified with qPCR before paired-end sequencing on an Illumina NextSeq 550 system (2 x 75 bp, performed by MAD: Dutch Genomics Service & Support Provider, Swammerdam Institute for Life Sciences, University of Amsterdam). Sequencing data were processed using the ATAC-Seq pipeline of the Kundaje lab (https://github.com/kundajelab/atac_dnase_pipelines) with default parameters and mouse genome version mm9.