title: Illumina HiSeq 4000 sequencing; 10x scRNA-seq analysis of a human milk cell sample compared to a non-lactating breast tissue sample
library_construction_protocol: Non-lactating human breast tissue was donated by participants undergoing elective aesthetic reduction mammoplasty. Single cell suspensions of non-lactating mammary cells were generated using an adaptation of a previously described protocol (Linnemann et al. 2015). Briefly, the freshly collected mammary tissue minced in digestion buffer supplemented with 1 µg/mL insulin which was added together with 800 U/mL collagenase and 100 U/mL hyaluronidase and mixed at 100rpm at 37°C for 3-4 hours. Following digestion, the resulting dissociated organoid fragments were washed twice in buffer and pelleted by 300g centrifugation for 5 minutes prior to cryopreservation.  On the day of library preparation, organoid fragments were gently defrosted in a 37°C water bath for approximately 5 minutes before being treated with Trypsin and dispase to yield a highly viable single cell suspension.  Human milk was freshly collected using a breast pump and diluted in an equal volume of sterile phosphate-buffered saline (PBS) and centrifuged at 870 g for 20 minutes at 20°C. The pellet was washed by removing the supernatant and resuspending in 5-10 mL of cold PBS and centrifuging at 490 g for 5 minutes at 4°C. Following a second wash with PBS, the single cell suspension was ready for library preparation. Approximately 10,000 lactation or 3x10,000 non-lactation derived mammary cells were loaded four separate channels of a single Chromium controller (10x Genomics) chip. Consequently, gel bead-in-emulsions (GEMS) were generated using 10X Genomics Single Cell 3' Reagent Kit v2. Sequencing libraries were prepared using Single Cell 3' Reagent Kits v2 (10X Genomics) and then converted using the NovaSeq 6000 S2 Reagent Kit (Illumina).