Illumina NovaSeq 6000 paired end sequencing; ATAC-seq of mouse luminal progenitors isolated from wild-type or Blg-Cre;Brca1f/f;p53+/- mouse models
ATAC-seq of mouse luminal progenitors isolated from wild-type or Blg-Cre;Brca1f/f;p53+/- mouse models
Lymph node divested mouse mammary glands were mechanically dissociated after collection and the finely minced tissue was transferred to DMEM/F12 (Gibco) + 10 mM HEPES (Gibco) + 2 mg ml-1 collagenase (Roche) + 200 U ml-1 hyaluronidase (Sigma) (CH) + gentamicin (Gibco) at 37 °C and vortexed every 30 min. After the lysis of red blood cells in NH4Cl, cells were briefly digested with warm 0.05% Trypsin-EDTA (Gibco), 5 mg ml-1 dispase  (Sigma)  and  1 mg ml-1 DNase  (Sigma)  and  filtered through a cell strainer (BD Biosciences). Mouse mammary cells were incubated in HF medium (Hank's balanced salt solution  (Gibco) + 1%  fetal  bovine  serum,  Gibco) + 10%  normal  rat  serum  (Sigma)  for 20 min on ice to pre-block. Mouse mammary cells were stained with the following primary antibodies:    Cd31-biotin  (eBioscience,  clone  390,  1 μg ml−1,    1:500);    Cd45-biotin (eBioscience, clone 30F11, 1 μg ml−1,  1:500);  Ter119-biotin  (eBioscience,  clone  Ter119, 1 μg ml−1,  1:500),  EpCAM-APC/Cy7 (Biolegend, clone G8.8, 0.5 μg ml−1,  1:500),  Cd49f-BV421 (Biolegend 313623, 2 μg ml−1,  1:100),  Cd49b-AF488 (Biolegend, clone HMα2, 1 μg ml−1, 1:500) and Sca1-AF647 (Biolegend, clone D7, 1 μg ml−1, 1:500). Cells were then stained  with  Streptavidin-PE/Cy7  (BD-Biosciences,  0.4 μg ml−1,  1:500).  Zombie  Aqua (Biolegend, 1:100) was used to detect dead cells. Human mammary cells were stained with the  following  primary  antibodies:  CD45-APC  (Biolegend,  clone  H130,1:100),  CD31-APC (Biolegend,  clone  WM-59,  1:100),  EPCAM-APC/Fire750  (Biolegend,  clone  9C4,  1:50), CD49f-PE/Cy7 (Biolegend, clone GoH3, 1 μg ml−1, 1:200). DAPI was used to detect dead cells. Cells were filtered through a cell strainer (Partec) before sorting. Sorting of cells was done using a FACS Aria Fusion sorter. Single-stained control cells were used to perform compensation manually. Unstained cells were used to set gates. After doublets, dead cells and contaminating haematopoietic, endothelial and stromal cells were gated out, mouse  CD49b+,  Sca1-luminal progenitors were sorted for ATAC-Seq experiments.  The tagmentation  reaction was performed on FACS-sorted luminal progenitors using the previously established protocol by Buenrostro et al (2013) https://doi.org/10.1038/nmeth.2688. Briefly, sorted luminal progenitor cells were spun down at 300g for 4 min at 4°C. Cell pellets were resuspended in cold 1mL 1xPBS and centrifuged again at 500g for 5 min at 4°C. Cells were lysed in 50μL of cold lysis buffer by gently pipetting samples up and down 12-16 times (10mM Tris-HCl (pH 7.4), 3mM MgCl2, 10mM NaCl and 0.1 (v/v) IGEPAL CA-630). Following cell lysis, resulting nuclei were spun down at 500g for 10 min at 4°C. Nuclei pellet was resuspended in 50μl of transposition mix (2.5μl 20x TDE1, 25μl 2x TD, 22.5μl H2O; Illumina Nextera 1502812) and incubated for 30 min at 37oC in a water bath. Tagmented DNA was purified using Qiagen MinElute PCR purification kit and eluted in 10μL EB buffer (10mMTris-Cl pH8.0).  ATAC-seq library preparation was performed by the NGS Facility at the Wellcome Trust Medical Research Council Stem Cell Institute using the Nextera DNA Library Prep Reference Guide. In general, libraries were amplified using 10μL of DNA, 2.5μL of i7 adapter, 2.5μL of i5 adapter, 7.5μL polymerase buffer mix and 2.5μL of primer mix. The PCR was run for 12 cycles with the following conditions: 72°C for 3 min, 98°C for 30 s; and thermocycling at 98°C for 10 s, 63°C for 30 s, 72°C for 3 min. Libraries were purified using an AMPure XP bead clean up with 1x volume.